Presentation of hydrophobic antigens to T-cells by CD1 molecules

ABSTRACT

Provided are CD-1 presented antigens, compositions, cells, inhibitors and methods relating to the use of hydrophobic antigen presentation by CD1 molecules, including: methods for detecting the presence of a CD1-presented hydrophobic antigen in a sample; methods for isolating such CD1-presented antigens and isolated antigens; vaccines containing CD1-presented antigens and vaccination methods; methods of blocking CD1 antigen presentation; methods of identifying and/or isolating CD1 blocking agents and the isolated CD1 blocking agents; methods of inducing CD1 expression; and T-cells for use in the methods disclosed herein.

RELATED APPLICATIONS

This application is a Divisional of U.S. application Ser. No.08/501,600, filed Jul. 12, 1995, now U.S. Pat. No. 6,238,676, which is aContinuation-in-part of U.S. application Ser. No. 08/322,980, filed Oct.13, 1994, now U.S. Pat. No. 5,679,347, which is a continuation of Ser.No. 08/322,979, filed Oct. 13, 1994, now U.S. Pat. No. 5,853,737, whichis a continuation-in-part of PCT International applicationPCT/US94/06991, designating the United States of America, and filed Jun.21, 1994, of which this application is a Continuation-in-part of U.S.application Ser. No. 08/080,072, filed Jun. 21, 1993, now abandoned,which is a Continuation-in-Part of U.S. application Ser. No. 07/989,790,filed Dec. 10, 1992, now abandoned, which patent applications areincorporated in their entirety herein by reference.

STATEMENT OF GOVERNMENT RIGHTS IN THE INVENTION

Part of the work performed during the development of the presentinvention utilized U.S. Government funds. The U.S. Government hascertain rights in the invention.

FIELD OF THE INVENTION

The invention disclosed herein is directed to CD1-presented antigens,compositions, cells, blocking agents and methods relating to the use ofhydrophobic antigen presentation by CD1molecules, including detection,isolation, vaccine production and administration, blocking of CD1presentation, blocking agents, induction of CD1 expression and isolatedT-cells. CD1-presented antigens of the invention stimulate T-cells toundergo an immune response.

DESCRIPTION OF THE BACKGROUND ART

The Immune System and T-cells

Animals have a complex array of molecular and cellular defenses,collectively referred to as the immune system, that recognize and attackpotentially harmful foreign or endogenous but abnormal cells(respectively represented by, e.g., pathogens such as bacteria orviruses, and cancerous or pathogen-infected cells), but tolerateendogenous normal cells. When stimulated by foreign or abnormalbiomolecules, the immune system undergoes a series of activitiesdesigned to neutralize and destroy the pathogens, or cancerous orpathogen-infected cells, with which the foreign or abnormal biomoleculesare associated. These activities, collectively known as an immuneresponse, may consist of a cell-mediated immune response, a humoral(antibody-mediated) immune response, or an immune response that includeselements of cell-mediated and humoral responses.

Humoral immune responses are mediated by antibodies that bind specificforeign or abnormal biomolecules. Antibodies are immunoglobulin (Ig)molecules produced by B-cells, as lymphocytes which originate in avianbursa or in mammalian bone marrow, but migrate to and mature in otherorgans, particularly the spleen. Robertson, M., Nature 301:114 (1983).Cell-mediated immune responses are the result of activities of T-cells,lymphocytes that undergo maturation within the thymus of an animal.Tizard, I. R., Immunology: An Introduction, 2d Ed., Saunders,Philadelphia (hereafter “Tizard”), p. 163, 1988. Both T and B-cellsmigrate between various organs and/or tissues within an animal's body.Lydyard, P., and Grossi, C., Chapter 3 in Immunology, 2d Ed., Roitt, I.,et al., eds., Gower Medical Publishing, London, New York, 1989.

T-cells mediate at least two general types of immunologic functions,effector and regulatory, reflecting the fact that T-cell activities varyconsiderably among different subpopulations of T-cells within an animal.Rook, G., Chapter 9 in Immunology, 2d Ed., Roitt, I., et al., eds.,Gower Medical Publishing, London, New York, 1989. Effector functionsinclude delayed hypersensitivity, allograft rejection, tumor immunity,and graft-versus-host reactivity. Effector functions reflect the abilityof some T-cells to secrete proteins called lymphokines, and the abilityof other T-cells (“cytotoxic” or “killer” T-cells) to kill other cells.The regulatory functions of T-cells are represented by the ability of“helper” T-cells. Helper T-cells interact with, and produce biomoleculesthat influence the behavior of, both B-cells and cytotoxic T-cells, inorder to promote and direct antibody production and cytotoxicactivities, respectively. Mosier, D. E., Science 158:1573–1575 (1967).Other classes of T-cells, including suppressor T-cells and memoryT-cells, also exist. Miedema, F., and Melief, C. J. M., Immunol. Today6:258–259 (1983); Tizard, pp. 225–228.

Antigen Recognition

In order to function properly, the T- and B-cells of an animal's immunesystem must accurately and reliably identify the enormous number ofmolecular compositions derived from foreign (“non-self”), or endogenous(“self”) but abnormally expressed, compositions that are encountered.Recognition and identification by the immune system occurs at themolecular level. An antigen, a molecular composition having thepotential to generate an immune response, is composed of one or moremolecular-sized identifying features known as epitopes. A polypeptideantigen which has an amino acid sequence which comprises, e.g., ahundred amino acids might comprise dozens of epitopes, wherein eachepitope is defined by a portion of the polypeptide comprising from about3 to about 25 amino acids. The number of eptitopes derivable frompolypeptides alone is estimated to be about ten million. Tizard, p. 25.

An antigen encountered by a T or B-cell of an animal must be identifiedas either being associated with normal endogenous (i.e., self) antigens,an immune response to which would be injurious to the animal, or withforeign or abnormal (i.e., non-self) antigens, to which an immuneresponse should be mounted. As part of the immune system's means ofidentifying antigens, individual T and B-cells produce antigen receptorswhich are displayed on the T or B-cell's surface and which bind specificantigens. Turner, M., Chapter 5 in Immunology, 2d Ed., Roitt, I., etal., eds., Gower Medical Publishing, London, New York, 1989. B-cellsproduce and display antigen receptors that comprise Ig molecules whichhave unique antigenbinding portions due to unique amino acid sequencesin the variable regions of each of the two antibody subunits, known asthe Ig heavy and Ig light chains. Each B-cell membrane comprises from20,000 to 200,000 identical Ig molecules. Tizard, pp. 78–80 and 202.

The T-cell antigen receptors (TCRs) produced by and displayed onindividual T-cells comprise heavy (TCRβ) and light (TCRα) chains(polypeptide subunits) which are linked by a disulfide bond on theT-cell surface. Each TCR α and β subunit has a carboxy-terminal constantregion, the amino acid sequence of which does not vary from T-cell toT-cell, and an amino-terminal variable region, the amino acid sequenceof which does vary from T-cell to T-cell. When TCRα and TCRβ subunitsassociate with each other, the variable regions of the TCRα and TCRβpolypeptide subunits combine to form the unique antigen-binding portionof an α:β TCR. A second type of TCR heterodimer, γ:δ, has been describedbut its function, if any, is unknown. Davis, M. M., and Bjorkman, P. J.,Nature 334:395–404 (1988). Although at least one mixed TCR heterodimerof unknown function, β:δ TCR, has been described, T-cells bearing α:βTCR molecules are numerically dominant in mature animals. Hochstenbach,F., and Brenner, M. B., Nature 340:562–565 (1989).

Although each individual T- or B-cell displays identical antigenreceptors, the receptor displayed varies from cell to cell; an animal'scollection of different antigen receptors is thus quite diverse. Thegenetic basis of this diversity is as follows. The variable region of anIg heavy chain, or that of a TCRβ chain, is encoded by three genesegments, the variable (V), diversity (D) and joining (J) segments. Thevariable region of an Ig light chain, or that of a TCRα chain, isencoded by V and J gene segments. Multiple DNA sequences encoding manydifferent V, D and J gene segments are present as unexpressed copies ingermline DNA; an analogous but different collection of variable genesegments for TCR subunits is also present. During development of ananimal, genes encoding diverse variable regions are generated inindividual cells of the immune system by the random joining of V, D andJ, or V and J, gene segments. The process of DNA rearrangements thatgenerates a randomly assembled variable region of an Ig heavy or TCRβsubunit is called V-D-J joining; the analogous process that generates arearranged variable region of an Ig light or TCRα subunit is called V-Jjoining. Sakano, H., et al., Nature 280:288–294 (1979); Early, P., etal., Cell 19:981–992 (1980); Alt, F. W., et al., Science 238:1079–1087(1987); Harlow, E., and Lane, D., Antibodies: A Laboratory Manual, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, pages 10–18, 1988;Davis, M. M., and Bjorkman, P. J., Nature 334:395–404 (1988).

A functionally rearranged Ig or TCR subunit gene is one in which the DNArearrangements of V-D-J or V-J joining have not resulted in a readingframe that is prematurely terminated because of the introduction of stopcodons or frameshifting mutations. Because each T or B-cell of theimmune system expresses genes encoding their respective antigenreceptors in which a unique functionally rearranged variable region ispresent, many different T or B-cells, each producing a receptor that hasa unique antigen-recognizing region, are generated. Hay, F., Chapter 6in Immunology, 2d Ed., Roitt, I., et al., eds., Gower MedicalPublishing, London, New York, 1989. The total catalog of differentantigen receptors displayed on the T-cells of an animal is referred toas the animal's TCR repertoire. Bevan, M. J., et al., Science264:796–797 (1994).

For mature T- or B-cells, binding of antigen to a cell's antigenreceptor activates the cell, i.e., stimulates the cell to undertakeactivities related to generating a cell-mediated or humoral immuneresponse. Typically, activated mature T or B-cells proliferate inresponse to antigen. In contrast, for immature T or B-cells, binding ofantigen to a displayed TCR or B-cell antigen receptor, respectively,results in elimination of the cell by a process called negativeselection or clonal deletion. Clonal deletion occurs during normaldevelopment of a healthy wildtype animal, and is a mechanism by whichthe immune system learns to tolerate the animal's normal endogenous(self) antigens, i.e., to treat the animal's self antigens asnon-immunogenic antigens. Failure of the immune system to achieve ormaintain tolerance of self antigens may result in autoimmune responses(i.e., autoimmune response to self antigens) that can culminate inautoimmune disease in animals including humans. Autoimmune disease canoccur when an appropriate immune response to a non-self antigen resultsin the production of immune effector biomolecules (e.g., autoantibodies)or cells that cross-react with self antigens. Human autoimmune diseasesinclude such crippling conditions as Multiple Sclerosis (MS) andSystemic Lupus Erythematosus (SLE). Roitt, I., Chapter 23 in Immunology,2d Ed., Roitt, I., et al., eds., Gower Medical Publishing, London, NewYork, 1989; Steinman, L., Sci. American 269:107–114 (1993).

Antigen Presentation

Although the antigen receptors of B-cells can directly bind solubleantigen, T-cells typically respond to antigen only when it is displayedon specific classes of other cells known generically as anantigen-presenting cells (APCs). Feldmann, M., and Male, D., Chapter 8in Immunology, 2d Ed., Roitt, I., et al., eds., Gower MedicalPublishing, London, New York, 1989. APCs, e.g., macrophages anddendritic cells, present antigens derived from polypeptides viaglycoproteins, known as MHC (major histocompatibility complex) proteins,which are displayed on the surface of APCs. Bevan, M. J., et al.,Science 264:796–797 (1994). The nomenclature for MHC gene productsvaries from species to species. For example, human MHC proteins are alsoreferred to as human lymphocyte antigens (HLA), murine MHC proteins arealso referred to as H-2 antigens, and rat MHC proteins are also calledRT1 antigens. Tizard, p. 181. Particular MHC proteins bind selectedclasses of antigens with limited specificity. For the most part, thespecificity determinants in a TCR:Ag:MHC complex are (1) the uniquepolypeptide sequences of the variable portion of the TCR and (2) theunique polypeptide sequences of antigen. However, to some degree,MHC-presented oligopeptide antigens are embedded within an MHC moleculeand TCR recognition of antigen only occurs within the context of anappropriate class of MHC molecule. Janeway, C. A., Sci. American269:73–79 (1993). This phenomenon, called MHC restriction, is offundamental importance to T-cell antigen recognition and physiology.Zinkernagel, R. M., and Doherty, P. C., Nature 248:701–702 (1974).

In MHC-mediated presentation of antigens, the α:β T-cell antigenreceptor recognizes peptide antigens in conjunction with products of MHCgenes. In the case of soluble antigens, recognition occurs inconjunction with Class II molecules. For viral antigens, recognition isin conjunction with Class I molecules. Furthermore, large solubleantigens are processed from polypeptides by an appropriate accessorycell, such as a macrophage or dendritic cell.

The general sequence of events involved in T-cell recognition ofpolypeptide antigens in MHC restriction is as follows. A polypeptideantigen is phagocytosed by an antigen-presenting cell, internalized,processed, and then a peptide derived from the polypeptide is displayedon the cell surface in conjunction with Class I or Class II MHCmolecules. In order to present antigen, MHC Class I molecules require anadditional protein, β₂-microglobulin. Tizard, pp. 181–183. A T-cellantigen receptor α:β heterodimer then recognizes the peptide antigenplus the MHC gene product. Recognition of peptide antigen alone or MHCgene product alone is not sufficient to signal T-cell activation. Onlythe MHC:Ag complex can be appropriately recognized by a TCR molecule.Steward, M., Chapter 7 in Immunology, 2d Ed., Roitt, I., et al., eds.,Gower Medical Publishing, London, New York, 1989.

The genes encoding MHC proteins are diverse; however, unlike Ig and TCRmolecules, which vary from cell to cell in an individual animal, MHCantigens vary from individual animal to individual animal or from onegroup of related individual animals to another group. Members offamilial groups, represented in the mouse by inbred strains of mice,share similar MHC antigens with each other, but not with individualsfrom other strains of mice. Snell, G. D., Science 213:172–178 (1981);Owen, M., Chapter 4 in Immunology, 2d Ed., Roitt, I., et al., eds.,Gower Medical Publishing, London, New York, 1989. Because variant MHCmolecules will be capable of binding different antigens, the antigensthat T-cells will be able to recognize (i.e., specifically bind in theMHC context) and respond to varies among different strains of mice.Cooke, A., Chapter 11 in Immunology, 2d Ed., Roitt, I., et al., eds.,Gower Medical Publishing, London, New York, 1989. In humans, particulargenetic alleles encoding MHC (HLA) molecules are more highly associatedwith autoimmune diseases, presumably because these MHC molecules aremore competent at binding (and thus presenting to T-cells) selfantigens. Vaughan, in Immunological Diseases, 3rd Ed., Vol. II, Samter,M., ed., pp. 1029–1037 (1978); Steinman, L., Sci. American 269:107–114(1993).

T-cell Subsets

Classes of T-cells are to some extent distinguished on the basis thatdifferent T-cells display different CD proteins on their surfaces.Immature T-cells display both CD4 and CD8 proteins (i.e., immatureT-cells are CD4⁺8⁺), mature helper T-cells are CD4⁺8⁻ (i.e., display CD4protein but not CD8 protein) and mature cytotoxic T-cells are CD4⁻8⁺(i.e., display CD8 protein but not CD4 protein). Smith, L., Nature326:798–800 (1987); Weissman, I. L., and Cooper, M. D., Sci. American269:65–71 (1993).

In most cases so far examined, CD8⁺ T lymphocytes recognize MHC class Icomplexes, while CD4⁺ cells recognize MHC class II complexes on antigenpresenting cells. The involvement of CD8 and CD4 in antigen recognitionby α:β TCRs is significant. CD4 and CD8 molecules increase the avidityof the TCR interaction Ag:MHC complexes and are sometimes referred to asco-receptors (Bierer, B. E., et al., Ann. Rev. Immunol. 7:579–599(1989); Steward, M., Chapter 7 in Immunology, 2d Ed., Roitt, I., et al.,eds., Gower Medical Publishing, London, New York, 1989). Because of theimportance of CD4 and CD8 in antigen recognition in the MHC context,CD4⁻8⁻ (double negative; DN) T-cells have classically been considered tobe immature thymic T-cell precursors. Lydyard, L., and Grossi, C.,Chapters 2 and 14 in Immunology, 2d Ed., Roitt, I., et al., eds., GowerMedical Publishing, London, New York, 1989; Smith, L., Nature326:798–800 (1987); Strominger, J. L., et al., Int. J. Cancer Suppl.4:43–47 (1989); Shirai, T., et al., J. Immunology 144:3756–3761 (1990);Weissman, I. L. and Cooper, M. D., Sci. American 269:65–71 (1993).

The DN subpopulation of T-cells is distinctive in regard to the TCRsthat they display. The majority of human DN T-cells isolated fromperipheral blood express δ:γ TCRs. Porcelli, S., et al., ImmunologicalReviews 120:137–183 (1991). A large proportion (approximately 60%) ofmurine DN α:β TCR T-cells express Vβ8 gene products (Fowlkes, B. J., etal., Nature 329:251–254 (1987); Bix, M., et al., J. Exp. Med.178:901–908 (1993)).

Several analyses in mice point to a striking lack of junctional (V-J orV-D-J) diversity and restricted use of germline V and J gene elements,especially for TCRα subunits. Koseki, H., et al., Proc. Natl. Acad. Sci.USA 87:5248–5252 (1990); Kubota, H., et al., J. Immunol. 149:1143–1150(1992). Examination of fresh DN α:β TCR T-cells in humans revealed astriking predominance of an invariant (canonical) Vα24-JαQ rearrangementthat lacked N region additions. Porcelli, S., et al., J. Exp. Med.178:1–16 (1993). Taken together, these observations suggest that DN α:βTCR T-cells may represent a developmentally distinct subpopulation of Tlymphocytes whose limited receptor repertoire reflects recognition of arestricted set of antigens and/or antigen-presenting molecules.

CD1 Proteins

Polypeptide molecules encoded by the genes of the CD1 locus arerecognized by select CD4⁻8⁻ T-cell clones expressing either α:β or γ:δTCRs (Porcelli, S., et al., Nature 341:447–450 (1989); Faure, F., etal., Eur. J. Immun. 20:703–706 (1990)). Because of the structuralresemblance of CD1 molecules, encoded by genes on human chromosome 1, toMHC molecules, encoded by genes on human chromosome 6 (Calabi, F. andMilstein, C., Nature 323:540–543 (1986); Balk, S. P., et al., Proc.Natl. Acad. Sci. USA 86:252–256 (1989)), it has been suggested that CD1may represent a family of antigen presenting molecules separate fromthose encoded by the MHC genes. Porcelli, S., et al., Nature 341:447–450(1989); Strominger, J. L., Cell 57:895–898 (1989); Porcelli, S., et al.,Immun. Rev. 120:137–183 (1991).

The five CD1 genes reveal exon and domain structure (α1, α2, α3) that issimilar to that of MHC class I genes, yet the proteins are onlydistantly related in sequence. All CD1 family members share a conservedα3 domain; however, even this domain shows only 32% homology in aminoacid sequence with consensus residues of class I MHC α3 domains andthere is no detectable homology with α1 domains. A major differencebetween MHC and CD1 molecules is polymorphism. Human MHC genes areextremely polymorphic: multiple alleles have been described at eachknown MHC locus. In contrast, CD1 genes are apparently nonpolymorphic.Despite these differences, the CD1 proteins, like MHC Class I molecules,are expressed as large subunits (heavy chains) non-covalently associatedwith β₂-microglobulin. Van Agthoven, A., and Terhorst, C., J. Immunol.128:426–432 (1982); Terhorst, C., et al., Cell 23:771–780 (1981)).

Five CD1 genes have thus far been identified in humans: CD1a, CD1b,CD1c, CD1d and CD1e. Four of the five CD1 gene products have beendefined serologically, are referred to as CD1a, CD1b, CD1c and CD1d andare distinguished by unique heavy chains with approximate molecularweights of 49 kDa, 45 kDa, 43 kDa and 48 kDa respectively (Amiot, M., etal., J. Immunol. 136:1752–1758 (1986); Porcelli, S., et al., Immunol.Rev. 120:137–183 (1991); Bleicher, P. A., et al., Science 250:679–682(1990)). CD1 proteins are displayed on a number of APCs includingLangerhans cells (which are the major dendritic antigen-presenting cellsin the skin), activated B-cells, dendritic cells in lymph nodes, and onactivated blood monocytes (Porcelli, S., et al., Nature 360:593–597(1992); Leukocyte Typing IV, Knapp, W., ed., Oxford University Press,Oxford, U.K., pp. 251–269, 1989; Tissue Antigens, Kissmeyer-Nielsen, F.,ed., Munksgard, Copenhagen, Denmark, pp. 65–72, 1989.

Previous work has shown that CD1 proteins are recognized by CD4⁻8⁻T-cell lines derived from patients with SLE. Porcelli, et al., Nature341:447–450 (1989). Leukemia cells expressing CD1 proteins were lysed bythe T-cells independent of MHC restriction, even though no foreign(non-self) antigen was present. DN T-cells lysed leukemic cells in aCD1-dependent manner in the absence of antigen. Thus, the possibilityexists that CD1 proteins play a role in autoimmune diseases.

The central dogma of immunology has been that the immune system does notnormally react to self. Autoimmunity defines a state in which thenatural unresponsiveness or tolerance to self terminates. As a result,antibodies or cells react with self constituents, thereby causingdisease. There is as yet no established unifying concept to explain theorigin and pathogenesis of the various autoimmune disorders. The diseaseprocess may be caused, among other things, by sensitized T lymphocytes.These lymphocytes produce tissue lesions by poorly understood mechanismswhich may involve the release of destructive lymphokines or whichattract other inflammatory cells to the lesion. For a review ofautoimmunity, see Theofilopoulos, A. N., Chapter 11 in Basic andClinical Immunology, 6th Ed., Stites, D. P., et al., eds., Appleton andLang, 1987.

Tuberculosis

Mycobacteria are a genus of aerobic intracellular bacterial organismswhich upon invasion of their host, survive within endosomal compartmentsof monocytes and macrophages. Human mycobacterial diseases includetuberculosis (caused by M. tuberculosis), leprosy (caused by M. leprae),Bairnsdale ulcers (caused by M. ulcerans), and various infections causedby M. marinum, M. kansasii, M. scrofulaceum, M. szulgai, M. xenopi, M.fortuitum, M. chelonei, M. haemophilum and M. intracellulare. Wolinsky,E., Chapter 37 in Microbiology: Including Immunology and MolecularGenetics, 3rd Ed., Harper & Row, Philadelphia, 1980, hereafter“Wolinksy”; Daniel, T. M., Miller, R. A. and Freedman, S. D., Chapters119, 120 and 121, respectively, in Harrison's Principles of InternalMedicine, 11th Ed., Braunwald, E., et al., eds., McGraw-Hill, New York,1987.

One third of the world's population harbors M. tuberculosis (M. tb.) andis at risk for developing tuberculosis (TB), which is specificallyresponsible for 18.5% of deaths in adults aged 15 to 59. Bloom, B. R.,and Murray, C. J. L., Science 257:1055–1064 (1992). Because improvedpublic health and antibiotic therapy have greatly reduced the occurrenceand/or severity of TB in the United States, these alarming statisticsderive largely from third-world countries. Unfortunately, with theadvent of AIDS, tuberculosis is increasing at a nearly logarithmic rate,and multidrug resistant strains are appearing and now account for onethird of all cases in New York City. Bloom, B. R., and Murray, C. J. L.,Science 257:1055–1064 (1992); U.S. Congress, Office of TechnologyAssessment, The Continuing Challenge of Tuberculosis, OTA-H-574, U.S.Government Printing Office, Washington, D.C., 1993. Mycobacterialstrains which were previously considered to be nonpathogenic strains(e.g., M. avium) have now become major killers of immunosuppressed AIDSpatients. Moreover, current Mycobacterial vaccines are eitherinadequate, in the case of the BCG vaccine to M. tb., or, with regard toM. leprae, unavailable. Kaufmann, S., Microbiol. Sci. 4:324–328 (1987);U.S. Congress, Office of Technology Assessment, The Continuing Challengeof Tuberculosis, pp. 62–67, OTA-H-574, U.S. Government Printing Office,Washington, D.C., 1993.

The major response to mycobacteria involves cell mediated delayedhypersensitivity (DTH) reactions with T-cells and macrophages playingmajor roles in the intracellular killing and containing or walling off(granuloma formation) of the organism. A major T-cell response involvesCD4⁺ lymphocytes that recognize mycobacterial heat shock proteins (suchas hsp65) as immunodominant antigens. Kaufmann, S. H., et al., Eur. J.Immunol. 17:351–357 (1987).

Leprosy

Leprosy (Hansen's disease) is a chronic granulomatous infection ofhumans which attacks superficial tissues, especially the skin andperipheral nerves. Accounts of leprosy extend back to the earliesthistorical records and document a stigmatization of leprosy patientswhich transcends cultural and religious boundaries. Miller, R. A.,Chapter 120 in Harrison's Principles of Internal Medicine, 11th Ed.,Braunwald, E., et al., eds., McGraw-Hill, New York, 1987, hereafter“Miller.” In ancient times leprosy was rampant throughout most of theworld, but for unknown reasons it died out in Europe in the sixteenthcentury and now occurs there only in a few isolated pockets. Wolinsky,p. 741.

There are probably 10 to 20 million persons affected with leprosy in theworld. The disease is more common in tropical countries, in many ofwhich the prevalent rate is 1 to 2 percent of the population. A warmenvironment is not critical for transmission, as leprosy also occurs incertain regions with cooler climates, such as Korea and central Mexico.Distribution of infected individuals within countries is verynonhomogeneous, and districts in which 20 percent of the population isaffected can be found. Miller, p. 633.

In the United States, leprosy occurs particularly in Texas, California,Louisiana, Florida, New York City, and Hawaii, usually in personsoriginally from Puerto Rico, the Philippines, Mexico, Cuba, or Samoa.Indigenous transmission occurs primarily in Hawaii, the Pacific Islandterritories, and specifically along the Gulf coast. Several hundredpatients are cared for at the national leprosarium in Carville, La.Wolinsky, p. 741.

Mycobacterium leprae, or Hansen's bacillus, is the causal agent ofleprosy. It is an acid-fast rod assigned to the family Mycobacteriaceaeon the basis of morphologic, biochemical, antigenic, and geneticsimilarities to other mycobacteria. M. leprae causes chronicgranulomatous lesions closely resembling those of tuberculosis, withepithelioid and giant-cells, but without caseation. The organisms in thelesions are predominantly intercellular and can evidently proliferatewithin macrophages, like tubercle bacilli. Wolinsky, p. 740.

Although M. leprae has not been cultivated in artificial media or tissueculture, it can be consistently propagated in the foot pads of mice.Systemic infections with manifestations similar to those of humandisease can be induced in armadillos and mangabey monkeys. The bacillusmultiplies exceedingly slowly, with an estimated optimal doubling timeof 11 to 13 days during logarithmic growth in mouse foot pads. The mousemodel has been used extensively for the study of antileprosy drugs, andthe high bacterial yield from armadillos has been crucial forimmunogenic studies. Miller, p. 633.

Leprosy is apparently transmitted when exudates of mucous membranelesions and skin ulcers reach skin abrasions; it is not highlycontagious and patients need not be isolated. Young children appear toacquire the disease on briefer contact than adults. The incubationperiod is estimated to range from a few months to 30 years or more.Apparently, M. leprae can lie dormant in tissues for prolonged periods.Wolinsky, p. 741. Leprosy can present at any age, although cases ininfants less than one year of age are extremely rare. The age-specificincidence peaks during childhood in most developing countries, with upto 20% of cases occurring in children under 10. Since leprosy is mostprevalent in poorer socioeconomic groups, this may simply reflect theage distribution of the high-risk population. The sex ratio of leprosypresenting during childhood is essentially 1:1, but males predominate bya 2:1 ratio in adult cases. Miller, p. 633.

Leprosy is distinguished by its chronic, slow progress and by itsmutilating and disfiguring lesions. These may be so distinctive that thediagnosis is apparent at a glance; or the clinical manifestations may beso subtle as to escape detection by any except the most experiencedobservers armed with a high index of suspicion. The organism has apredilection for skin and for nerve. In the cutaneous form of thedisease, large, firm nodules (lepromas) are distributed widely, and onthe face they create a characteristic leonine appearance. In the normalform, segments of peripheral nerves are involved, more or less atrandom, leading to localized patches of anesthesia. The loss ofsensation in fingers and toes increases the frequency of minor trauma,leading to secondary infections and mutilating injuries. Both forms maybe present in the same patient.

In either form of leprosy, three phases may be distinguished. (1) In thelepromatous or progressive type, the lesions contain many lepra cells:macrophages with a characteristically foamy cytoplasm, in whichacid-fast bacilli are abundant. When these lesions are prominent, thelepromin test is usually negative, presumably owing to desensitizationby massive amounts of endogenous lepromin, and the cell-mediated immunereactions to specific and nonspecific stimuli are markedly diminished.The disease is then in a progressive phase and the prognosis is poor.(2) In the tuberculoid or healing phase of the disease, in contrast, thelesions contain few lepra cells and bacilli, fibrosis is prominent, andthe lepromin test is usually positive. (3) In the intermediate type ofdisease, bacilli are seen in areas of necrosis but are rare elsewhere,the skin test is positive, and the long-range outlook is fair. Shiftsfrom one phase to another, with exacerbation and remission of thedisease, are common.

Hansen's bacillus may be widely distributed in the tissues of personswith leprosy, including the liver and spleen. Nevertheless, nodestructive lesions or disturbance of function are observed in theseorgans. Most deaths in leprous patients are due not to leprosy per sebut to intercurrent infections with other microorganisms—oftentuberculosis. Leprosy itself often causes death through the complicationof amyloidosis, which is characterized by massive waxy deposits,containing abundant precipitates of fragments of immunoglobulin lightchain in kidneys, liver, spleen, and other organs. Wolinsky, pp.740–741.

Bacteriologic diagnosis of leprosy is accomplished by demonstratingacid-fast bacilli in scrapings from ulcerated lesions, or in fluidexpressed from superficial incisions over non-ulcerated lesions. Nouseful serologic test is available, but patients with leprosy frequentlyhave a false-positive serologic test for syphilis. Also useful in thetuberculoid phase is the skin test with lepromin, an antigenic bacillarymaterial prepared by boiling human lepromatous tissue or infectedarmidillo tissues, which is typically standardized to contain 160×10⁶acid-fast bacilli/ml. Wolinsky, pp. 740–741.

Therapy with dapsone (4,4′-diaminodiphenylsulfone) or related compoundsusually produces a gradual improvement over several years, and iscontinued for a prolonged period after apparent clinical remission.However, resistance to sulfonic drugs, with a concomittant relapse, maybe noted after years of apparently successful treatment. Rifampin andclofazimine (B663, a phenozine derivative) are promising agents nowunder investigation for treating leprosy. Treatment results may beevaluated by counting the acid-fast bacilli in serial biopses and skinscrapings. Wolinsky, p. 741.

Because of the neurodegenerative nature of leprosy, and associatedsymptoms, the management of leprosy involves a broad, multidisciplinaryapproach, including consulatative services such as orthopedic surgery,opthamology, and physical therapy in addition to antimicrobialchemotherapy. In any event, however, recovery from neurologic impairmentis limited. Miller, pp. 635–636.

SUMMARY OF THE INVENTION

The present invention is based on the novel and unexpected observationthat CD1 molecules function to present foreign antigens as well asautoimmune antigens to T-cells. The invention is further based on theobservation that isolated blood monocytes can be induced to express CD1,and therefore become competent to present antigens to T-cells, bycontacting the monocytes with cytokines. Based on these twoobservations, the present invention discloses methods of isolating CD1⁺antigen-presenting cells (CD1⁺ APCs) which are used to identify,isolate, and purify CD1-presented hydrophobic antigens, various methodsfor determining whether a sample contains one or more CD1-presentedantigens, methods for isolating and purifying CD1-presented antigens,purified CD1-presented antigens isolated by the methods disclosedherein, and methods of providing and using isolated CD1-presentedantigens in vaccines.

In one embodiment, the present invention provides methods fordetermining whether a sample contains a CD1-presented hydrophobicantigen. In one such method, the presence of a CD1-presented antigen inthe sample can be determined by (1) contacting the sample with cellswhich have been induced to express a CD1 protein, (2) contacting thecells from the first step with T-cells that specifically recognize aCD1-presented antigen, and (3) measuring the proliferative or cytolyticresponse of the T-cells, wherein increased T-cell proliferation orT-cell-mediated cytolysis of CD1⁺ target-cells, respectively, correlateswith the presence of a CD1-presented antigen. In a related embodiment,the present invention provides for methods of determining whether asample contains a CD1 blocking agent, i.e., a composition that inhibitsCD1-restricted antigen presentation. In the related embodiment, theassay for CD1-presented antigen described above is performed induplicate, with a first (control) assay being performed as above, and asecond assay additionally containing a sample suspected of containing aCD1 blocking agent. The presence of CD1 blocking agents in the samplecorrelates with a T-cell proliferative or cytolytic response in thesecond assay that is less than that measured in the first assay.

The present invention further provides methods for inducing CD1expression in cells, such as monocytes, in order to generate CD1⁺antigen-presenting cells (APCs). In one method, CD1 expression isinduced in isolated blood monocytes by contacting the cells with one ormore cytokines. The preferred cytokines for CD1 induction aregranulocyte/macrophage colony stimulating factor (GM-CSF), GM-CSF incombination with interleukin-4 (IL-4), or interleukin-3 (IL-3). CD1⁺APCs are cells that express and display CD1 proteins and are thuscompetent to present CD-restricted antigens to TCR⁺ T-cells. CD1⁺ APCsare used in several of the methods disclosed herein.

The present invention further provides methods for isolating aCD1-presented antigen from a sample. In one such method, a samplecontaining a CD1-presented antigen is first fractionated usingconventional techniques. The resulting fractions are then tested usingknown procedures, or those disclosed herein, for the presence of aCD1-presented antigen. The fractions containing the CD1-presentedantigen are then either used in the development of vaccines or arefurther fractionated to obtain higher levels of purity of theCD1-presented antigen.

The present invention further provides alternative methods for isolatingCD1-presented antigens from a sample which rely on the ability of aCD1-presented antigen to bind either isolated CD1 or CD1 expressed on acell surface. In one such method, a sample containing a CD1-presentedantigen is incubated with either CD1⁺ APCs or purified CD1 molecules.The resulting complexes of antigen:CD1⁺ APC or antigen: CD1 molecule arethen removed from the sample and subjected to conditions in which theCD1 molecule releases the bound CD1-presented antigen. The releasedCD1-presented antigen is then purified away from either the CD1⁺ APC orthe purified CD1 molecule and may be further characterized usingconventional immunological, biochemical and/or genetic methods. PurifiedCD1-presented antigens, or synthetic or genetically engineeredderivatives thereof, are then tested for CD1-presented antigen activityusing known procedures, or those disclosed herein, and may be used inthe formulation of vaccines.

Utilizing the above procedures for isolating a CD1-presented antigen,the present invention further provides isolated CD1-presented antigenswhich have been prepared by the methods disclosed herein. The isolatedCD1-presented antigens prepared by the disclosed methods can be usedeither in the characterization of the nature of CD1-presented antigens,in the development or formulation of vaccines, or in the development ofautoimmune therapies.

The present invention is further based on the observation thatCD1-mediated antigen presentation can serve as a basis for thedevelopment of autoimmune disease. Based on this observation, thepresent invention provides methods of and means for inhibitingCD1-mediated antigen presentation by a CD1⁺ APC. CD1-mediated antigenpresentation can be inhibited by various compositions that are describedherein or are isolated by the methods of the invention.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A–H show data for the expression of CD1a, CD1b and CD1c bymonocytes cultured with GM-CSF and IL-4, and surface phenotype ofCD1b-restricted T-cells specific for Mycobacterium tuberculosis. Flowcytometric analysis of peripheral blood monocytes cultured for 60 hoursin medium containing GM-CSF and IL-4 showing expression of CD1a (FIG.1A), CD1b (FIG. 1B), CD1c (FIG. 1C), and HLA (FIG. 1D). Cells werestained with control monoclonal antibody (mAb) (dotted line) or mAbswith the specificity indicated in each histogram box (solid lines).Monocytes cultured in the absence of cytokines or with interferon-γ didnot express significant levels of CD1a, CD1b or CD1c (data not shown).Flow cytometric analysis of T-cell line DN1 showing its expression ofα:β TCRs (FIG. 1E), non-expression of CD4 (FIG. 1F), and minimal ornon-expression of CD8 (FIGS. 1G, 1H). Dotted and solid lines representcontrol and specific mAbs as in FIGS. 1 a–1 d.

FIGS. 2A–E show data for antigen specificity and self restriction ofproliferative responses of CD4⁻8⁻ T-cell line DN1 and its subcloneDN1.C7. FIG. 2A shows data for the proliferative responses (counts perminute (CPM)) of ³H-thymidine incorporated) of DN1 to M. tuberculosis(solid squares), M. leprae (solid circles), Escherichia coli (opencircles) and tetanus toxoid (open squares). Antigen presenting cellswere heterologous GM-CSF- and IL-4-treated CD1⁺ monocytes. Antigenconcentration (based on protein content) is shown on the x-axis. FIG. 2Bshows data for the proliferative response of T-cell line DN1 to M.tuberculosis (1 μg protein/ml) requires CD1⁺ antigen presenting cells(CD1⁺ APCs). APCs indicated by symbols as follow: no APCs, open square;GM-CSF and IL-4 treated monocytes (CD1⁺ APCs), closed circles; IFNγtreated monocytes (CD1⁺), open circles; freshly isolated monocytes(CD1⁺), open triangles. The number of APCs added to each culture isshown on the x-axis. FIG. 2C shows data indicated that APC's from alldonors tested supported the proliferative response of T-cell line DN1 toM. tuberculosis. Open bars, T-cells plus APCs without M. tuberculosis;solid bars, T-cells plus APCs with M. tuberculosis (1 μg protein/ml).APCs were GM-CSF and IL-4 treated peripheral blood mononuclear cellsfrom five unrelated donors. HLA typing confirmed that no allele of theHLA-A, -B, -C, -DR, -DP or -DQ loci was shared among all five donors(data not shown). FIGS. 2D, 2E show data indicating that anti-CD1b mAbspecifically inhibited the proliferative response of DN1 (FIG. 2D) andDN1.C7 (FIG. 2E) to M. tuberculosis (1 μg protein/ml). APCs were GM-CSF-and IL-4-treated monocytes. Solid bars, proliferative response ofT-cells to APCs with M. tuberculosis (1 μg protein/ml); dotted lines,response to APCs in the absence of M. tuberculosis; “nd, ” notdetermined. Monoclonal antibodies used were P3 (control IgG), OKT6(anti-CD1a), WM-25 (anti-CD1b; Favaloro, E. J., et al., Disease Markers4:261–270(1986)), 10C3 (anti-CD1c), W6/32 (anti-MHC Class I), and IVA12(anti-MHC Class II; Shaw, S., Hum. Immun. 12:191–211 (1985)).

FIG. 3 show data for a comparison of the ability of antigen presentingcell lines CR1 and cytokine stimulated monocytes to stimulate growth ofT-cell lines 2.13.DN1 and G7, clones derived from T-cell line DN1. Openbars, T-cells plus APCs without M. tuberculosis; solid bars, T-cellsplus APCs with M. tuberculosis (1 μg protein/ml).

FIGS. 4A–D show data for the presentation of M. tuberculosis by CD1transfectants of the lymphoblastoid cell line C1R. C1R cells stablytransfected with vector pSRα-NEO DNA (mock) or with constructs ofpSRα-NEO containing cDNAs encoding the indicated CD1 molecule (CD1a,CD1b and CD1c) were cultured for 12 hours in medium alone (open bars) orin medium containing M. tuberculosis (25 μg protein/ml, filled bars),labeled with ⁵¹Cr and used as target-cells for cytolytic assay withvarious effector T-cells. The effector T-cell to target-cell ratio was50:1. FIG. 4A. shows for M. tb. CD1b-presented Ag-specific T-cell lineDN1. FIG. 4B shows data for DN1 subclone DN1.C7. FIG. 4C shows data forCD1a autoreactive clone BK6. FIG. 4D shows data for CD1c autoreactiveclone 3C8.

FIGS. 5A–C shows data indicating that CD1b restricted presentation of M.tuberculosis antigen does not require MHC Class II region encodedmolecules, but does involve antigen processing by a chloroquinesensitive pathway. FIG. 5A shows data for lysis of CD1 T2 transfectantsby T-cell line DN1. T2 cells transfected with vector DNA alone (mocktransfectant) are indicated by circles, and T2 cells transfected withCD1b by triangles. Open symbols represent target-cells not preincubatedwith M. tuberculosis, and filled symbols represent target-cellspreincubated for 12 hours with M. tuberculosis (10 ug protein/ml). Flowcytometric analysis showed that incubation of CD1b transfected T2 cellswith M. tuberculosis had no effect on CD1b expression (data not shown).FIG. 5B shows data indicating that glutaraldehyde fixation of CD1b⁺ APCsprevents presentation of M. tuberculosis to line DN1. CD1b⁺ APCs(GM-CSF- and IL-4-treated peripheral blood mononuclear cells, PBMCs)were cultured for 12 hours in the presence of M. tuberculosis (1 μgprotein/ml; “Pulsed APCs”) or in medium alone (“Unpulsed APCs”),harvested and an aliquot of each cell suspension was fixed with 0.0125%glutaraldehyde for 30 seconds. The resulting APC preparations weretested for their ability to stimulate proliferation of line DN1 in theabsence (open bars) or presence (solid bars) of soluble M. tuberculosisantigen (1 μg protein/ml). FIG. 5C shows data for inhibition of CD1brestricted presentation of M. tuberculosis by chloroquine. CD1b⁺ APCsfrom an HLA-DR7⁺ individual were pulsed with M. tuberculosis antigen for60 minutes at 37° C. in the presence of the indicated concentration ofchloroquine, fixed with glutaraldehyde, and used as APCs inproliferative assays with line DN1 (solid circles) or with the M.tuberculosis specific, HLA-DR7⁺ restricted CD4⁺ T-cell line DG.1 (opentriangles). Results are expressed as percent inhibition of responsescompared to fixed APCs pulsed with M. tuberculosis in the absence ofchloroquine, and are representative of three similar experiments.

FIG. 6 shows data indicating the effect on the proliferative response ofT-cell line DG.1 to M. tuberculosis antigen of digestion of antigen withthe indicated proteases.

FIG. 7 shows data for the effect on the proliferative response of T-cellline DN1 to M. tuberculosis antigen of digestion of antigen with theindicated proteases.

FIG. 8 shows data for the effect on the proliferative response of T-cellline DN1 to M. fortuitum antigen of digestion of antigen with theindicated proteases.

FIGS. 9A–C shows data indicating that a Mycobacterial antigen recognizedby a DN α:β TCR⁺ T-cell line quantitatively partitions into the organicphase after extraction with organic solvents and is CD1b restricted.Extraction with organic solvents differentiates the CD1b-restrictedMycobacterial antigen from Mycobacterial antigens recognized by aconventional MHC class II restricted CD4⁺ α:β TCR⁺ T-cell line and thesmall nonprotein Mycobacterial ligand recognized by DN γ:δ (Vγ2Vδ2) TCR⁺T-cells. Pfeffer, K., et al., J. Immunology 148:575–583 (1992). TotalMycobacterial sonicates were extracted with chloroform/methanol/H₂O andthe resultant three phases were assayed by culturing T-cells with CD1⁺monocytes and the indicated dilutions of the various antigenpreparations. FIG. 9A shows data for the proliferative response of theCD1b-restricted DN T-cell line DN1 to total mycobacterial sonicates (▪,dashed line), organic phase (□, solid line), aqueous phase (◯, solidline) or interface (▪, solid line). Antigen concentration along the xaxis is depicted as 1/dilution normalized to the standard total sonicatepreparation. FIG. 9B shows data for the proliferative response of theHLA-DR7 (MHC) restricted Mycobacterial specific CD4⁺ T-cell line DG.1 toMycobacterial fractions after extraction with organic solvents. FIG. 9Cshows data for the proliferative response of the Vγ2Vδ2 T-cell cloneDG.SF68 to Mycobacterial fractions after extraction with organicsolvents.

FIG. 10 shows the cytolytic response of the DN1 line to CD1transfectants of C1R cells pulsed with Mycobacterial antigenpreparations. CD1b or CD1c transfectants (Porcelli, S., et al., Nature341:447–450 (1989)) of C1R lymphoblastoid cells were used as targets ina standard cytolytic assay pulsed either with M. tuberculosis antigenpreparations after extraction with organic solvents (+) or media alone(−). Recognition by the T-cell line DN1 of C1R cells transfected withCD1b occurs only when pulsed with antigen. No antigen specificrecognition occurs for CD1c⁺ targets.

FIG. 11 shows the chemical structure of 6,6-trehalose dimycolate (cordfactor).

FIGS. 12A–E shows data indicating that the Mycobacterial antigenrecognized by the CD1b-restricted T-cell line DN1 is mycolic acid. FIGS.12A, 12B show data indicating that the proliferative response of theCD1b-restricted T-cell line DN1 correlates with mycolic acid peaks onreverse phase C18 HPLC. The purified Mycobacterial acyl chain fractioncontaining all the CD1b-restricted antigen was chromatographed usingreverse phase HPLC and the resulting fractions assayed for the abilityto stimulate a proliferative response by the T-cell line DN1. FIG. 12Ashows data for display of the absorbance spectrum at 254 angstrom(expressed as optical density units, OD, ×10⁻⁴) (solid line) of theeluted material and the corresponding methylene chloride concentration(dotted line) of the elution gradient. The large absorbance peak elutingbetween 2 to 6 minutes is free bromophenacyl bromide, the derivitizingagent. FIG. 12B shows data for the proliferative response of the T-cellline DN1 to each one minute fraction. The CD1b-restricted antigenresponse is seen as a broad peak correlating with mycolic acid. FIG. 12Cshows data indicating that saponified 6,6-trehalose dimycolate (cordfactor), but not saponified trehalose dibehenate, stimulates aproliferative response by the CD1b restricted T-cell line DN1. Mycolicacids were generated by saponification of purified trehalose dimycolatefrom either M. tuberculosis (H37Ra) or M. kansasii. Trehalose dibehenate(synthetic cord factor) was treated in an identical fashion. Antigenconcentration is expressed in μg/ml of cord factor along the x axis.FIGS. 12D, 12E show data for the reversed phase HPLC analysis ofpurified trehalose dimycolate from M. tuberculosis (H37Ra) results inthe stimulation of the CD1b-restricted T-cell line DN1 by fractionscorresponding to mycolic acid peaks. The saponified trehalose dimycolateof M. tuberculsis was chromatographed as in the experiment shown in FIG.12A (FIG. 12D), and fractions assayed for the ability to induce aproliferative response by the line DN1 (FIG. 12E). As in FIG. 12A,bioactivity correlates with early mycolic acid peaks.

FIG. 13 shows data for the cytolytic response of the DN1 T-cell line toCD transfectants of C1R cells pulsed with mycolic acid prepared from M.tb. cord factor (Sigma) by saponification. CD1a, CD1b, CD1c or mocktransfectants of C1R lymphoblastoid cells were pulsed with mycolic acidsprepared from trehalose dimycolate (+) or media alone (−) and used astargets in cytolytic assays, the results of which are given as %specific lysis.

FIGS. 14A–C shows data indicating that mycolic acid is not mitogenic,but a specific antigen restricted by CD1b and recognized by the T-cellline DN1. Four T-cell lines specific for Mycobacteria and two additionalT-cell lines were tested for the ability to respond to either total M.tuberculosis sonicates, mycolic acid preparations from purified cordfactor or HPLC purified mycolic acids from either M. tb. sonicates orcord factor. The responses of three representative Mycobacterialspecific T-cell lines are shown, DN1 (▪) (DN, CD1b-restricted, α:βTCR⁺), DG.1 (□) (CD4⁺, HLA-DR7 restricted, α:β TCR⁺) and DN6 (◯) (DN,CD1c-restricted, α:β TCR⁺. APCs for all six T-cell lines tested wereidentically GM-CSF- and IL-4-treated (CD1⁺) PBMCs from an HLA-DR7positive individual. FIG. 14A (upper panel) shows data for theproliferative responses of three Mycobacterial specific T-cell lines tototal sonicates of M. tb. (H37Ra, Sigma). Antigen concentration isdisplayed on the x axis as cpm×10⁻³. The three T-cell lines shown allrespond to total Mycobacterial sonicates. FIG. 14B (middle panel) showsdata for the proliferative response to HPLC-purified mycolic acidsisolated from M. tb. sonicates. Only the CD1b-restricted T-cell line DN1responds to purified mycolic acid. FIG. 14C (bottom panel) shows datafor the proliferative responses to HPLC-purified mycolic acids generatedfrom purified M. tb. cord factor (Sigma). Only the CD1b-restrictedT-cell line DN1 proliferates in response to cord factor mycolic acids.Not shown are three additional T-cell lines tested in the sameexperiment, SP-F3 (Roncarlo, M. G., et al., J. Exp. Medicine168:2139–2152 (1988)) (CD4⁺ α:β TCR⁺, DR restricted, tetanus toxoidspecific), CP.1.15 (Morita, C. T., et al., Eur. J. Immunol. 21:2999–3007(1991)) (DN, Vγ2Vδ2 TCR⁺, Mycobacterial specific), BK6 (Porcelli, S.,Nature 341:447–450 (1989) (DN, α:β TCR⁺, autoreactive to CD1a). Allthree did not respond to purified mycolic acids, but two proliferated inresponse to their specific antigen (tetanus toxoid—SP-F3, <1 kDa M.tuberculosis preparation—CP.1.15). BK6 exhibits cytolytic activityagainst CD1a, but is unable to proliferate in response to CD1a⁺ APCs ofany type tested. Porcelli, S., Nature 341:447–450 (1989).

FIGS. 15A–B shows data indicating the effect of the indicated monoclonalantibodies on the proliferative response of T-cell line 2.13.DN1 (FIG.15A) and 8.23.DN1 (FIG. 15B).

FIGS. 16A–D shows data for CD1c-restricted presentation of M.tuberculosis antigen to T-cell line DN2. The results of cytolytic assaysof CR1 cells transfected with vector (mock, FIG. 16A) and with DNAmolecules encoding the indicated CD1 protein (CD1a, CD1b and CD1c)(FIGS. 16B, 16C, 16D, respectively), wherein the transfected cells wereeither preincubated with (filled circles) or without (open circles) M.tuberculosis.

FIGS. 17A–D shows data for CD1c-restricted presentation of M.tuberculosis antigen to T-cell line DN6. The results of cytolytic assaysof CR1 cells transfected with vector (mock, FIG. 17A) and with DNAmolecules encoding the indicated CD1 protein (CD1a, CD1b and CD1c)(FIGS. 17B, 17C, 17D, respectively), wherein the transfected cells wereeither preincubated with (filled circles) or without (open circles) M.tuberculosis.

FIG. 18 shows data for the prliferative response of the CD1c-restrictedcell line DN6 to M. tb. antigens in sonicates after extraction of theantigens with organic solvents. Proliferation is in cpm (³H thymidineincorporation) displayed on the y axis. APCs were CD1 expressingmonocytes. Antigens were titered over 6 logs and the results from arepresentative point (1:3,750 dilution of antigen) are shown. Backgroundcpm (defined from a media alone control) were subtracted from allvalues.

FIG. 19 shows data for the proliferative response of the CD1c-restrictedcell line DN6 to M. tb. antigens in sonicates before and aftersaponification of the antigens. Proliferative response in cpm isdisplayed on the y axis and the concentration of antigen (shown as1/dilution) is displayed on the x axis. The equivalent of 10 mg of M.tb. (strain H37Ra; Difco) was sonicated in PBS and was either useddirectly or first saponified. All antigen dilutions were normalized tothe standard initial concentration of 200 mg lyophilized bacteria in 5ml.

FIGS. 20A–B shows data indicating that DN α:β TCR⁺ T-cell lines fromleprous skin lesions are CD1-restricted. FIG. 20A (upper panel) showsdata indicating that anti-CD1c mAb specifically inhibited theproliferative response of T-cell line LDN1 to M. leprae. FIG. 20B (lowerpanel) shows data indicating that anti-CD1b mAb specifically inhibitedthe proliferative response of T-cell line LDN4 to M. leprae.

FIG. 21 shows data for the proliferative response of T-cell line LDN4 tothe indicated cellular fractions of M. leprae.

FIG. 22 shows the chemical structure of lipoarabinomannan (LAM).Abbreviations: Manp=mannopyranose; Araf=arabinofurnaose.

FIG. 23 shows data indicating that the T-cell line LDN4 responds to LAMin a CD1b-restricted manner.

FIG. 24 shows data for the response of T-cell line LDN4 to LAMderivatives. Abbreviations: dLAM=dacylated LAM; PIM=phosphatidylinositolmannoside.

FIG. 25 shows data indicating that the T-cell line BDN2 responds to LAMfrom M. leprae (Lep LAM) as well as LAM from a clinical isolate of M.tuberculosis (TBE LAM) and a virulent laboratory strain of M.tuberculosis (Rv LAM).

FIGS. 26A–B shows data for the flow cytometric analysis of OGD1 and OAB8T-cell lines. OGD1 T-cells were stained with biotin-conjugated anti-γδTCR monoclonal antibody TCRδ1 or anti-Vδ1 monoclonal antibody δTCS1(revealed with PE-streptavidin), (FIG. 26A). OAB8 T-cells were labelledwith anti-αβ TCR monoclonal antibody BMA031 (followed byPE-streptavidin), anti-CD4-FITC monoclonal antibody OKT4,anit-CD8-α-chain-FITC monoclonal antibody OKT8 or anti-CD8-β-chain-FITC2ST8-5H7 (FIG. 26B).

FIG. 27 shows data indicating that the TCR γδ⁺ T-cell line OGD1 respondsto Mycobacterial antigens which partition in the organic phase of achloroform/methanol extraction. For the organic fraction, whole PBSsonicates of Mycobacteria tuberculosis (DIFCO, Detroit, Mich.) (200 mgbacteria/5 ml PBS) were extracted (4:1, v/v organic to aqueous) withchloroform:methanol (2:1, v/v). Cord factor was obtained from SIGMAchemicals (St. Louis, Mo.). To prepare free mycolic acid, cord factorwas saponified (25% KOH in methanol: H₂O 1:1, 121° C., 1 hr.) andmycolic acids extracted into hexane. Preparations were dried with NO₂and resuspended in media/fetal calf serum and tested at differentdilutions in standard proliferation assay (50,000 activated monocyteswith 50000 T-cells in a 3 day assay).

FIG. 28 shows data indicating that the TCR γδ⁺ T-cell line OGD1 isrestricted by CD1b, as demonstrated by cytotoxic assays of the TCR γδ⁺T-cell line on antigen pulsed activated monocytes. To prepare targetcells, the monocytes were incubated overnight with no antigen (openboxes) or the organic fraction (filled boxes). Target cells were thenlabelled with ⁵¹Cr and T-cells were added to the target cells at a ratioof 10:1 with different blocking antibody (no monoclonal antibodies,anti-MHC I (BB7.7)), mixture of all anti-CD1 monoclonal antibodies,anti-CD1a (OKT6), anti-CD1b (WM25) or anti-CD1c (10C3)). The measurementof the cytotoxicity was calculated as in standard cytotoxic assays,i.e., specificcytolysis=(Experimental−Spontaneous)/(Maximum−Spontaneous)×100.

FIG. 29 shows data indicating that the CD8⁺ αβ⁺ T-cell line OAB8responds to Mycobacterial mycolic acid. Mycolic acid was purified byHPLC from M. tb. (prepared as described in Example 4) and used at 1μg/ml. Control antigens are Diphtheria toxoid (available from Dr. R.Finberg, Dana Farber Cancer Institute, Boston, used at 10 μg/ml) and M.tb. LAM (as in Example 8; 1 μg/ml). Fractions were resuspended inmedia/fetal calf serum and tested as described in FIG. 27.

FIG. 30 shows data indicating that the CD8⁺ αβ⁺ T-cell line OAB8 isrestricted by CD1c, as demonstrated by proliferation assays performed asin FIG. 27. Monoclonal antibodies were added to the culture at a 1/200final dilution of ascites. Antigens are as described in FIG. 29 andantibodies are the same as in FIG. 28.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Glossary

Antigen: A molecule, compound or composition of matter which (1) inducesan immune response in an animal, alone or in combination with anadjuvant; and (2) interacts specifically with one or moreantigen-recognizing components of the animal's immune system.

Foreign antigen: An antigen which is not endogenous to a normal, healthyanimal.

Autoimmune antigen: An endogenous molecule, compound or composition ofmatter in an animal which comprises an antigen in an autoimmune disease.This term is synonymous with “self antigen” and “autoantigen.”

CD1-presented antigen: An antigen which is bound by a member of the CD1family of proteins and displayed on the surface of an CD1⁺ APC.CD1-presented antigens vary in their size and composition depending ontheir origin and the member of CD1 family that they are recognized by.As used herein, the term “CD1-presented antigen” includes those antigensidentified herein and/or those antigens isolated using the knownprocedures or those disclosed herein. This term is synonymous with“CD1-restricted antigen.”“CD1-bound antigen” designates a CD1-presentedantigen that is bound to its appropriate CD1 molecule.

Hydrophobic antigen refers to any hydrophobic molecule or composition,containing amino acids, sugars or lipids, or any combination thereof,which contains some hydrophobic portion.

CD1 family of proteins: A collection of proteins which have beenidentified by their structure, immunologic cross-reactivity and/ordistribution, as being related to known CD1 molecules. A specific CD1protein may be referred to as a member of the CD1 family of proteins.Members of the CD1 family of proteins include, but are not limited to,CD1a, CD1b, CD1c, CD1d and CD1e (see, Porcelli, S., et al., Immun. Rev.120:137–183 (1991)).

CD1 positive cell: A cell which expresses and displays one or moremembers of the CD1 family of proteins. This term is synonymous with“CD1⁺ cell.” One skilled in the art can use the procedures describedherein, or known in the art, for determining whether a cell isexpressing one or more members of the CD1 family of proteins (seeExample 1 and Porcelli, S., Immun. Rev. 120:137–183 (1991)).

Antigen-presenting cell (APC): An APC includes any cell which displaysantigen molecules on its surface via protein carriers and which presentsantigen to T-cells. Antigen-binding protein carriers include MHC class Imolecules, MHC class II molecules and CD1 molecules; corresponding APCsare designated MHC I⁺ APCs, MHC II⁺ APCs and CD1⁺ APCs.

CD1-restricted T-cell: A mature TCR positive (TCR⁺) T-cell which canrecognize a CD1-bound CD1-presented antigen. CD1-restricted T-cellsinclude any subset of T-cells which interact with a CD1-boundCD1-presented antigen.

CD4⁻8⁻ T-cell: A mature TCR⁺ T-cell which does not express CD4 and CD8.This term is synonymous with “double negative T-cell” and “DN T-cell.”Techniques for identifying CD4⁻8⁻ T-cells are well known in the art andcan readily be employed in the present invention, for example using flowcytometry as described in Example 1, and/or in Panchomoorthy, G., etal., J. Immuno. 147:3360–3369 (1991)). Using such procedures, threeCD4⁻8⁻ T-cell lines, designated DN1, DN2 and DN6, have been isolated andare described herein.

Adjuvant: A molecule, compound or composition of matter which, whenintroduced into an animal with an antigen, enhances one or more immuneresponses to that antigen.

Genetic engineering: This term refers to any human manipulation intendedto introduce genetic change.

Sample: Any solution, emulsion, suspension, or extract which can betested using known procedures, or those disclosed herein. A sample maybe, but is not limited to, a soluble extract or an organic extract.Examples 1 and 2 provide various non-limiting types of samples derivedfrom Mycobacterium tuberculosis.

Contacting: The process of incubating or placing in proximity one itemwith another. For example, when a cell is contacted with a sample, thecell is incubated with the sample.

Fractionating: Subjecting a sample to conditions or procedures whichseparate the components of the sample based on physical or chemicalproperties such as, but not limited to, size, charge, solubility, orcomposition. Examples of fractionation procedures include, but are notlimited to, selective precipitation, organic extraction, size exclusiondialysis or chromatography, and ion exchange chromatography.

Expressing: The process of producing a product involving transcriptionof a DNA molecule to generate a corresponding mRNA molecule that isoptionally translated into a polypeptide by ribosomes and associatedcellular factors.

Displaying: The process of localizing a protein, or a protein:antigencomplex, to the outermost surface of a cell where the protein orprotein:antigen complex is accessible to a second cell or to moleculesdisplayed by a second cell. A protein, or a protein-antigen complex, issaid to be displayed by a cell when it is present on the outermostsurface of the cell and is thus accessible to a second cell and/or tomolecules displayed by a second cell.

Processing of antigen: The process by which an antigen is treated bycellular factors in order to be made competent for displaying.

CD1 blocking agent: A composition or compound which is capable ofblocking the interaction of a CD1-presented antigen with CD1, or ofblocking the interaction between CD1:antigen complexes and their cognateT-cell receptors. Blocking agents include (1) agents which bind to CD1,(2) agents which bind to a CD1-presented antigen, (3) agents which bindto a CD1:antigen complex, (4) agents which bind to a T-cell receptorthat recognizes a CD1: antigen complex and (5) agents which prevent theprocessing of a CD1-presented antigen.

The Present Invention

The present invention is based on the unexpected observation that CD1molecules function to present hydrophobic antigens to T-cells. Theinvention is further based on the observation that cells can be inducedto express CD1, and therefore become competent to present antigens toT-cells. Such presentation can involve contacting the cells withcytokines such as, but not limited to, granulocyte/macrophage colonystimulating factor (GM-CSF) and interleukin-4 (IL-4).

Based on these two observations, the present invention comprises:

-   -   methods for detecting the presence of a CD1-presented        hydrophobic antigen in a sample;    -   methods for isolating such CD1-presented antigens and the        isolated antigens;    -   vaccines containing CD1-presented antigens and vaccination        methods;    -   methods of blocking CD1 antigen presentation;    -   methods of identifying and/or isolating CD1 blocking agents and        the isolated CD1 blocking agents;    -   methods of inducing CD1 expression; and    -   T-cells for use in the methods disclosed herein.

In one embodiment, the present invention provides methods fordetermining whether a sample contains a CD1-presented antigen. In onesuch method, the presence of a CD1-presented antigen in a sample can bedetermined by: first, contacting the sample with a CD1 positive cell;second, contacting the cell of the first step with a T-cell; and third,measuring the proliferation of the T-cell.

Methods of characterizing classes of T-cells, and of isolatingsubpopulations of T-cells, are known. See, e.g., Wysocki, L. J., andSato, V. L., Proc. Natl. Acad. Sci. (USA) 75:2844–2848 (1978); Wasik, M.A., and Morimoto, C., J. Immunol. 144:3334–3340 (1990); Harriman, G. R.,et al., J. Immunol. 145:4206–2414 (1990); Koulova, L., et al., J.Immunol. 145:2035–2043 (1990); Steward, M., and Male, D., Chapter 25 inImmunology, 2d Ed., Roitt, I., et al., eds., Gower Medical Publishing,London, New York, 1989.

Methods of culturing T-cells in vitro, and of immortalizing T-cells viafusion to non-growth restricted cells such as myelomas, are also known.See, e.g., Paul, W. E., et al., Nature 294:697–699 (1981); Williams, N.,Nature 296:605–606 (1982).

Techniques for identifying T-cells subsets that are reactive with CD1presented antigens are also known in the art and can readily be employedin the present invention, for example using flow cytometry as describedin the Examples and/or by known techniques such as those descibed inPanchomoorthy, G., et al., J. Immuno. 147:3360–3369 (1991)). The presentinvention advances these techniques by providing methods for enrichingT-cell populations to obtain isolated T-cell clones which are reactiveto CD1-presented antigens. For example, a population of T-cells isallowed to divide and a subpopulation of mixed T-cells is isolated basedon proliferation in the presence of CD1⁺ APCs and CD1-presented antigen,or on cytolytic activity against transfected cells expressing CD1molecules in the presence of a CD1-presented antigen. Using suchprocedures, non-limiting examples of such CD1 reactive T-cell subsetshave been identified, including CD4⁻8⁻ T-cell lines, CD8⁺ TCR αβ⁺ T-celllines, and TCR γδ⁺ T-cell lines.

The present invention further provides methods for inducing CD1expression on a cell. In one such method, a cell can be induced toexpress CD1 by contacting the cell with one or more cytokines, as knownin the art. The preferred cytokines for CD1 induction aregranulocyte/macrophage colony stimulating factor (GM-CSF), GM-CSF incombination with interleukin-4 (IL-4), or interleukin-3 (IL-3). Example1 discloses that monocytes can be induced to express various members ofthe CD1 family by contacting the monocyte with 100 units each of GM-CSFand IL-4 for 60 hours in RPMI-1640 supplemented with 10% fetal calfserum. Using the methods and materials disclosed herein, one skilled inthe art can readily vary the contacting time, cytokine type, cytokineconcentration, and contacting conditions to obtain favorable results solong as the contacting step is sufficient to induce CD1 expression.

Several procedures are known in the art for determining theproliferation of T-cells and can be used in the above methods. Oneskilled in the art can readily adapt such procedures for use in thepresent invention. One such procedure, described in Example 1, measuresthe rate of incorporation of ³H-thymidine via liquid scintillation andby methods described in Morita, C. T., et al., Eur. J. Immunol.21:2999–3007 (1991)).

The present invention further provides methods for isolating aCD1-presented antigen from a sample. In one such method, a sample isfirst fractionated using conventional procedures. The fractions of thesample are then tested for the presence of a CD1-presented antigen asoutlined above. Examples 2 and 3 describes fractionation proceduresusing organic extraction with chloroform:methanol and silicic acidchromatography to fractionate a sample containing an extract of M.tuberculosis to purify a CD1-presented antigen.

The present invention further provides methods for isolating aCD1-presented antigen which rely on the specificity of binding of CD1 toa CD1-presented antigen. In one such method, a sample containing aCD1-presented antigen is first contacted with either purified CD1, or acell which expresses and displays CD1 (a “CD1⁺ cell”). The resultingantigen: CD1 complex, or antigen: CD1⁺ cell complex, is then separatedfrom the sample. Using such a procedure, a purified antigen: CD1complexor antigen: CD1⁺ cell complex is obtained. To further purify theCD1-presented antigen, either type of complex is treated underappropriate conditions such that the CD1-bound antigen will be releasedfrom the CD1 molecule.

The above two isolation methods can be combined by the skilled artisanto derive other methods for isolating CD1-presented antigens. In onesuch combination, a sample is fractionated, as described above, prior toperforming a purification method which relies on the binding of aCD1-presented antigen to CD1.

The present invention further provides CD1-presented antigens which areidentified or isolated using known procedures, or those disclosedherein. Unlike MHC-presented antigens, CD1-presented antigens are notlimited to polypeptides. One CD1-presented nonpeptide antigen, describedin detail in Examples 2–4, is a lipid antigen isolated from M.tuberculosis that comprises mycolic acids. Another CD1-presented antigenfrom M. tuberculosis, described in Examples 5 and 6, is a more complexlipid. A CD1-presented antigen from M. leprae is lipoarabinomannan(LAM), described in Examples 7–9. CD1-presented antigens are hydrophobicand have use in vaccine formulation and development.

The CD1-presented antigens of the present invention, i.e., thoseidentified or isolated using known procedures, or those disclosedherein, are readily usable as vaccines. A skilled artisan can employroutine formulation procedures in order to formulate an isolatedCD1-presented antigen for use as a vaccine. In addition to the CD-1presented antigen in such vaccines, the vaccine can further comprise atleast one additional CD-1 molecule or at least one MHC-I or MHC-IIpresented antigen. See, e.g., Remington's Pharmaceutical Sciences, 18thEd., Gennaro, A. R., ed., Mack, Easton, 1990; The Pharmacologist Basisof Therapeutics, 7th Ed., Gilman, A. G., et al., eds., MacMillian, NewYork, 1985; Avery's Drug Treatment: Principles and Practice of ClinicalPharmacology and Therapeutics, 3rd Edition, ADIS Press, Ltd., Williamsand Wilkins, Baltimore, Md. (1987).

The CD1-presented antigens of the present invention can be purified asdisclosed herein over a wide range of purities. A skilled artisan willknow how to employ various purification strategies in order to obtain aCD1-presented antigen which has been purified to the extent required foran intended use.

The vaccines of the present invention can be formulated using a purifiedCD1-presented antigen or can be formulated using a CD1-bound antigen.Because CD1-restricted antigens are presented to T-cells as a complex ofantigen and CD1, the use of an antigen:CD1 complex can, in some cases,provide superior immunization properties.

The present invention further provides methods for assaying forinhibitors of CD1-restricted antigen presentation to T-cells, i.e., CD1blocking agents. In one such method, CD1 antigen presentation isinhibited by using a CD1 blocking agent to block the ability of aCD1-restricted antigen to bind to CD1. As used herein, a CD1 blockingagent is said to “inhibit CD1-restricted antigen presentation” when theCD1 blocking agent decreases (1) the binding of a CD1-presented antigento a CD1 molecule or (2) the binding of a CD1:CD1-presented antigencomplex to its cognate T-cell receptors. Some CD1 blocking agents areable to block such binding to undetectable levels while other CD1blocking agents only slightly decrease such binding. CD1 blocking agentsinclude, but are not limited to, (1) agents which bind to CD1, (2)agents which bind to the CD1-presented antigen, (3) agents which bind tothe CD1:antigen complex, and (4) agents which bind to the T-cellreceptors that recognize the CD1:antigen complex. Respective examples ofblocking agents include, but are not limited to, (1) polyclonal ormonoclonal antibodies which bind to and block the portion of a CD1molecule that binds a CD1-presented antigen, (2) polyclonal ormonoclonal antibodies which bind to and block the portion of aCD1-presented antigen that binds CD1, (3) synthetic oligopeptides thatare derived from the CD1:antigen-binding portion of a T-cell receptorand which bind to and block the portion of the CD1:antigen complex boundby intact T-cell receptors, and (4) synthetic compounds comprising aCD1-presented antigen chemically linked to a purified CD1 molecule or asynthetic derivative thereof.

In an alternative method for inhibiting antigen presentation ofCD1-restricted antigens, a CD1 blocking agent can be employed whichblocks the interaction of the antigen:CD1 complex with the TCR moleculeson the T-cell. By inhibiting the presentation step, the activation ofspecific subsets of T-cells can be inhibited.

Pilot trials are currently underway of treatment of humans sufferingfrom an autoimmune disease with peptides derived from TCR molecules.Oksenberg, J. R., et al., J. Neurol. Sci. 115 (Suppl.):S29-S37 (1993).

DNA molecules encoding TCR polypeptides displayed by T-cells thatrecognize the CD1-presented antigens of the invention are isolatedaccording to methods known in the art. Oskenberg, J. R., et al., Proc.Natl. Acad. Sci. (USA) 86:988–992 (1989); Oksenberg, J. R., et al.,Nature 345:344–346 (1990) and erratum, Nature 353:94 (1991); Uematsu,Y., et al., Proc. Natl. Acad. Sci. (USA) 88:534–538 (1991); Panzara, M.A., et al., Biotechniques 12:728–735 (1992); Uematsu, Y., Immunogenet.34:174–178 (1991).

The DNA sequence is converted into a polypeptide sequence, and theportion of the polypeptide sequence that corresponds to theantigen-binding variable region of a TCR polypeptide is used to designsynthetic oligopeptides that bind CD1:antigen complexes on APCs, therebyinhibiting antigen presentation.

Oligopeptides are chemically synthesized according to standard methods(Stewart and Young, Solid Phase Peptide Synthesis, Pierce Chemical Co.,Rockland, Ill., 1985) and purified from reaction mixtures by reversedphase high pressure liquid chromatography (HPLC).

Additionally or alternatively, methods for generating anti-TCRantibodies and anti-TCR binding peptides are well known in the art withregard to MHC presentation and can readily be adapted to the hereindisclosed CD1 presentation system. Strominger, J. L., Cell 57:895–898(1989); Davis, M. M., and Bjorkman, P. J., Nature 334:395–404 (1989).

A skilled artisan can readily employ known methods of antibodygeneration, as well as rational blocking agent design in order to obtainthe blocking agents of the present invention. Harlow, E., and Lane, D.,Antibodies: A Laboratory Manual, Cold Spring Harbor Press, Cold SpringHarbor, 1988; Synthetic Peptides: Answers Guide, Freeman, W. H., NewYork, 1991; Kasprzak, A. A., Biochemistry 28:9230–9238 (1989).Additionally or alternatively, libraries of molecularly diversemolecules can be screened for individual member molecules which are CD1blocking agents. Effective CD1 blocking agents are identified by theirability to inhibit CD1-mediated T-cell proliferative and/or cytolyticresponses using the materials and methods described herein.

The embodiments of the invention described above can be used for theindicated purposes, alone or in combination with each other or othercomplementary methods and/or compositions.

The manner and method of carrying out the present invention may be morefully understood by those of skill by reference to the followingexamples, which examples are not intended in any matter to limit thescope of the present invention or of the claims directed thereto.

EXAMPLE 1 Antigen Presentation by CD1b

Methods

Flow cytometry was performed as described previously (Panchamoorthy, G.,et al., J. Immunology 147:3360–3369 (1991)) using the followingmonoclonal antibodies (mAbs): P3 (IgG1 control; Panchamoorthy, G., etal., J. Immunology 147:3360–3369 (1991)), OKT6 (anti-CD1a; Reinherz, E.,et al., Proc. Natl. Acad. Sci. (USA) 77:1588–1592 (1980)), 4A7.6(anti-CD1b; Olive, D., et al., Immunogenetics 20:253–264 (1984)), 10C3(anti-CD1c; Martin, L. H., et al., Proc. Natl. Acad. Sci. (USA)84:9189–9193 (1987)), W6/32 (anti-HLA-A,B,C; Brodsky, F. M., and Parham,P. P., J. Immunology 128:129–135 (1982)), BMA031 (anti-α:β TCR; Lanier,L. L., et al., in Leukocyte Typing III, McMichael, A. J., ed., pp.175–178, Oxford University Press, 1987), OKT4 (anti-CD4; Reinherz, E.,et al., Proc. Natl. Acad. Sci. (USA) 77:1588–1592 (1980)), OKT8(anti-CD8α; Reinherz, E., et al., Proc. Natl. Acad. Sci. (USA)77:1588–1592 (1980)) and 2ST8-5H7 (anti-CD8β; Shiue, L., et al., J. Exp.Med. 168:1993–2005 (1988)).

Monocytes were isolated from leukocyte concentrates of normal donors byplastic adherence (Anegon, I., et al., J. Immunology 147:3973–3980(1991)), and detached by incubation at 37° C. in phosphate bufferedsaline (PBS) with 0.53 mM EDTA (PBS/EDTA). Adherent-cells weretypically >90% CD14⁺ and MHC class II⁺, and negative for CD1a, CD1b andCD1c as determined by surface staining (data not shown). To induce CD1expression, monocytes were cultured for 60 hours in RPMI-1640 (Gibco)containing 10% fetal calf serum (FCS, Hyclone) with 100 Units/ml each ofGM-CSF and IL-4 (Genetics Research Institute). Cells were harvestedusing PBS/EDTA as above.

T-cell line DN1 was established from a random normal donor's peripheralblood. Nonadherent mononuclear cells were treated with mAbs 0KT4 andOKT8 and rabbit complement, and the remaining viable cells weresuspended in a mixture of 0KT4 (anti-CD4), 0KT8 (anti-CD8α) andanti-TCRδ1 (Porcelli, S., et al., Immun. Rev. 120:137–183 (1991)) mAbsfor 1 hour, washed and incubated for 30 minutes at 4° C. with goatanti-mouse immunoglobulin coupled magnetic beads (Dynal). After magneticseparation and removal of CD4⁺ and/or CD8⁺ and/or δ-TCR cells, theremaining CD4⁻8⁻ α:β TCR⁺ cells were cultured with equal numbers ofautologous monocytes in complete medium (RPM-1640 with 10% FCS andadditional supplements as previously described by Morita, C. T., et al.(Eur. J. Immun. 21:2999–3007 (1991)) with 100 U/ml each of GM-CSF andIL-4. M. tuberculosis soluble extract, produced by sonication ofdesiccated bacilli (strain H37Ra (Difco)) in PBS followed bycentrifugation at 100,000 g to clarify (i.e., to remove insolublematerial from) the sonicates, was added to a bacterial proteinconcentration of 10 μg/ml. More soluble antigenic (i.e., T-cellproliferative) activity is obtained from soluble aqueous sonicates of M.tb. by adding detergents such as CHAPS or octylglucoside during thesonication step; without addition of detergent, 90 to 95% of antigenicactivity is lost during post-sonification clarification. Cultures wererestimulated every 10 to 14 days with M. tuberculosis and heterologousCD1⁺ monocytes (induced to express CD1 as described above) in completemedium, and were fed every three to four days with fresh mediumcontaining 1 nM recombinant interleukin-2 (IL-2).

T-cell proliferation response assays were carried out in triplicate with5×10⁴ each of T-cells and irradiated (5,000 Rad) APCs in 200 μl completemedium in 96 well flat bottom microtiter plates (Linbro). M. leprae andEscherichia coli soluble extracts were produced as described for M.tuberculosis. Monoclonal antibodies were added as purifiedimmunoglobulin to a final concentration of 25 μg/ml. Cultures wereharvested on day five (day three for mAb blocking) after a six hourpulse with 1 μCi ³H-thymidine (6.7 Ci/mmol, New England Nuclear), and ³Hincorporation determined by liquid scintillation counting. Results areexpressed as the mean counts per minute (CPM) of ³H-thymidineincorporation of triplicate cultures. Isolated monocytes or wholeperipheral blood mononuclear cells (PBMCs) were treated with recombinantGM-CSF and IL-4 as above, or with IFN-γ 100 U/ml for 60 hours prior tocombining them with T-cells for proliferation assays. T-cell cloneDN1.C7 is representative of four extensively characterized subclonesderived from DN1 by phytohemagglutinin (PHA) stimulation in limitingdilution culture and propagated using PHA stimulation and IL-2 aspreviously described. Morita, C. T., et al., Eur. J. Immun. 21:2999–3007(1991). All clones derived from line DN1 had a surface phenotypeindistinguishable from that shown in FIG. 1 b, i.e., α:β TCR expression,non-expression of CD4, and minimal or non-expression of CD8.

T-cell cytolytic response assays were carried out as follows. Methods oftransfecting C1R cells and assaying specific cytolytic activity by ⁵¹Crrelease have been described. Balk, S. P., et al., Science 253:1411–1415(1991) and Morita, C. T., et al., Eur. J. Immun. 21:2999–3007 (1991),respectively. BK6, an α:β TCR cytotoxic T-cell clone that lyses cellsdisplaying CD1a, was isolated from the blood of a patient with SLE aspreviously described (Porcelli, S., et al., Nature 341:447–450 (1989)),and clone 3C8, an α:β TCR cytotoxic T-cell clone that lyses cellsdisplaying CD1c, was isolated from the blood of a normal donor using thesame method. Transfected cells were labeled with ⁵¹Cr and used astarget-cells in cytolytic assays with an effector (T-cell) to target(transfectant) ratio of about 50:1. Methods of assaying ⁵¹Cr release andcalculating the % specific lysis have been described. Brenner, M. B., etal., Nature 325:689–694 (1987).

Stable transfectants of T2 cells were prepared using the methoddescribed for C1R cells. Balk, S. P., et al., Science 253:1411–415(1991). APCs for glutaraldehyde fixation and chloroquine experimentswere GM-CSF- and IL-4-treated PBMCs as described above, andglutaraldehyde fixation and chloroquine treatment of APCs were performedaccording to published methods. Chesnut, R. W., et al., J. Immun.129:2382–2388 (1982); Roncarolo, M. G., et al., J. Immunol. 147:781–787(1991). CD4⁺ T-cell line DG.1 was derived from an HLA-DR7⁺ rheumatoidarthritis patient by repeated stimulation of synovial fluid lymphocyteswith autologous EBV-transformed B-cells and M. tuberculosis purifiedprotein derivative (PPD, Statens Serum Institute; data not shown).Proliferative response assays were performed as above, except that 2×10⁵APCs were added per well and ³H-thymidine incorporation was determinedafter three days.

Results

In order to develop a system to detect antigen presentation by CD1molecules, the ability of various recombinant cytokines to induceexpression of CD1a, CD1b and CD1c on peripheral blood monocytes, whichnormally do not express significant levels of these molecules, wasassessed. Leukocyte Typing IV, Knapp, W., ed., Oxford University Press,Oxford, U.K., pp. 251–269, 1989. High levels of CD1a, CD1b and CD1c wereconsistently observed on monocytes cultured with a combination ofgranulocyte/monocyte colony stimulating factor (GM-CSF) andinterleukin-4 (IL-4) (FIG. 1 a). Alternatively, GM-CSF may be usedalone, although the resultant level of CD1 expression is somewhat lessthan that resulting from combined GM-CSF and IL-4 treatment.Interleukin-3 (IL-3) may also be used, alone or in combination withother cytokines. Monocytes cultured in the absence of cytokines, orthose cultured with interferon-γ, did not express significant levels ofC1Da, CD1b, or CD1c (data not shown).

Because monocytes are efficient antigen presenting cells (APCs), wereasoned that CD1⁺ monocytes might stimulate a CD1 restricted T-cellresponse to an exogenous antigen. Because most CD1 specific T-cellsidentified to date have a double negative (DN; CD4⁻8⁻) phenotype(Porcelli, S., et al., Nature 341:447–450 (1989); Faure, F., et al.,Eur. J. Immun. 20:703–706 (1990)), we focused on this subset of cellsand generated a T-cell line by repeated stimulation of peripheral bloodof α:β TCR⁺ CD4⁻8⁻ T-cells with a soluble extract of M. tuberculosis andheterologous CD1⁺ monocytes (FIG. 1 b).

Functional studies of the resulting T-cell line (designated DN1) showedthat these T-cells gave specific proliferative responses to antigensderived from M. tuberculosis and from closely related M. leprae bacilli,but not to unrelated bacterial antigens such as those from E. coli ortetanus toxoid (FIG. 2 a). These responses were dependent on themonocytes being pretreated with GM-CSF and IL-4 (FIG. 2 b), and were notrestricted by polymorphic MHC determinants (FIG. 2 c). This lack of MHCrestriction was consistent with antigen presentation restriction bynon-MHC molecules. In order to determine if CD1 molecules are requiredfor M. tuberculosis antigen presentation, the effects of monoclonalantibodies (mAbs) specific for CD1 or MHC molecules on the M.tuberculosis induced proliferation of T-cell line DN1 and arepresentative subclone, DN1.C7, were determined. Only anti-CD1b mAbshowed significant blocking of the M. tuberculosis induced proliferativeresponse, and no consistent effects were observed with anti-CD1a or CD1cmAbs or with mAbs against monomorphic determinants of MHC class I orclass II molecules (FIG. 2 d).

B-cell transfectants are effective targets for α:β TCR CD4⁻8⁻ cytolyticT-cell activity. Using the B lymphoblastoid cell line C1R (Zenmour, J.,et al., J. Immun. 148:1941–1948 (1992)), stable transfectants expressingand displaying CD1a, CD1b or CD1c at comparable levels were generatedand tested for their ability to present M. tuberculosis in a cytolyticassay. Only C1R cells transfected with DNA sequences encoding CD1b andincubated with M. tuberculosis prior to the assay were lysed by α:β TCRDN T-cell line DN1 and its subclone DN1.C7 (FIGS. 4 a and b). Thespecificity of this CD1b restricted response was confirmed using twocontrol CD4⁻8⁻ α:β TCR⁺ T-cell clones, BK6 and 3C8, which were derivedby mitogen stimulation without exposure to M. tuberculosis antigens.Previous studies show that BK6 and 3C8 lyse target-cell lines expressingCD1a and CD1c, respectively (data not shown). As for all other CD1reactive T-cell clones described prior to this disclosure (Porcelli, S.,et al., Nature 341:447–450 (1989); Faure, F., et al., Eur. J. Immun.20:703–706 (1990); Balk, S. P., et al., Science 253:1411–1415 (1991)),these clones appear to be autoreactive and recognize theirnonpolymorphic CD1 ligands in the absence of exogenous antigens. Asexpected, clones BK6 and 3C8 lysed only C1R transfectants expressingCD1a or CD1c respectively, and lysis was not significantly affected byprior incubation of the target-cells with M. tuberculosis (FIGS. 4 c andd).

The lack of MHC restriction demonstrated by the preceding experimentsargued that MHC encoded antigen presenting molecules were not involvedin the CD1b restricted presentation of M. tuberculosis antigens to lineDN1. As a more stringent test of this hypothesis, CD1b transfectants ofthe T2 cell line, in which extensive chromosomal deletions in both MHCloci result in a complete lack of MHC class II molecule expression, wereproduced. Salter, R. D., et al., Immunogenetics 21:235–246 (1985);Erlich, H., et al., Hum. Immun. 16:205–219 (1986). The MHC linkedtransporter genes TAP-1 and TAP-2 (reviewed in Parham, P., Nature357:193–194 (1992)) are also deleted from T2, resulting in defectiveexpression and function of MHC class I molecules. Hosken, N. A., andBevan, M., Science 248:367–370 (1990); Wei, M., and Cresswell, P.,Nature 356:443–446 (1992). Nevertheless, transfection of CD1b into T2led to expression of CD1b on the cell surface at a level similar to thatseen on other transfected B-cell lines (data not shown) and generated atarget-cell that presented M. tuberculosis to line DN1 (FIG. 5 a).

The presentation of exogenous antigens to T-cells generally requiresuptake and processing of complex protein antigen molecules by antigenpresenting cells, a process which is blocked by aldehyde fixation of theAPC surface and by lysosomotropic amines such as chloroquine. Ziegler,H. K., and Unanue, E. R., Proc. Natl. Acad. Sci. USA 79:175–179 (1982);Chesnut, R. W., et al., J. Immun. 129:2382–2388 (1982). By thesecriteria, CD1b restricted presentation of M. tuberculosis also showed arequirement for antigen uptake and processing. Mild fixation of CD1b⁺APCs with glutaraldehyde completely abrogated their ability to stimulateline DN1 in the presence of M. tuberculosis soluble antigens, althoughthe same APCs pulsed with M. tuberculosis prior to fixation retainedtheir ability to stimulate a proliferative response (FIG. 5 b).Furthermore, the presentation of M. tuberculosis antigens to line DN1was strongly inhibited by chloroquine with a dose dependence virtuallyidentical to that for the inhibition of MHC class II mediatedpresentation of mycobacterial antigens (FIG. 5 c), indicating thatprocessing of antigens for CD1b and MHC class II restricted responsesmay involve similar pathways or organelles, or that the pathways shareone or more chloroquine-sensitive cellular factors. Interestingly, T2cells have recently been shown to be defective in the processing ofantigens presented by MHC class II molecules (Riberdy, J. M., andCresswell, P., J. Immun. 148:2586–2590 (1992)) because T2 cells lack theDMA and DMB genes. Morris, P., et al., Nature 368:551–554 (1994); Fling,S. P., et al., Nature 368:554–558 (1994). Thus, our finding that CD1btransfected T2 cells can present M. tuberculosis to DN1 suggests thatthe antigen processing requirements for CD1b and MHC class II molecules,although similar with regard to chloroquine sensitivity, are notidentical.

Several investigators have speculated that T-cells lacking expression ofboth CD4 and CD8 molecules may recognize antigens presented by cellsurface molecules other than those encoded by classical MHC class I andII loci. Porcelli, S., et al., Immun. Rev. 120:137–183 (1991); Janeway,C. A. Jr., et al., Immun. Today 6:73–76 (1988); Bluestone, J. A., andMatis, L. A., J. Immun. 142:1785–1788 (1989). The above results showthat one member of the CD1 family, CD1b, can restrict the specificresponse of MHC unrestricted CD4⁻8⁻ T-cells to an exogenous foreignantigen. Like other CD1 proteins, CD1b heavy chains associatenoncovalently with β₂-microglobulin (Olive, D., et al., Immunogenetics20:253–264 (1984)) and show limited but significant sequence homology toboth MHC class I and class II molecules. Calabi, F., and Milstein, C.,Nature 323:540–543 (1986); Balk, S. P., et al., Proc. Natl. Acad. Sci.USA 86:252–256 (1989). These structural features of CD1b, together withits critical role in antigen recognition (described above), support theconclusion that CD1b is a nonpolymorphic antigen presenting moleculeencoded by a genetic locus unlinked to the MHC.

These results indicate a potential role for CD1 restricted T-cells innormal host defense against microbial disease. The above results suggesta functional parallel between CD1 and MHC class II molecules, since bothmediate presentation of exogenous antigens processed through achloroquine sensitive pathway, and both can also act as ligands for theTCRs of autoreactive T-cells. Porcelli, S., et al., Nature 341:447–450(1989); Glimcher, L. H., and Shevach, E. M., J. Exp. Med. 156:640–645(1982). The limited tissue distribution of CD1 molecules in vivoprovides a further similarity with the MHC class II family since membersof both families are prominently expressed on cell types involved inantigen presentation to T-cells, including Langerhans's cells, dendriticcells in lymphoid and many other tissues, 8 cells and possibly cytokineactivated monocytes. Porcelli, S., et al., Immun. Rev. 120:137–183(1991). In contrast, the lack of structural polymorphism of CD1molecules, their unique cytokine regulation on monocytes, and the CD4⁻8⁻phenotype of the CD1 restricted T-cells described herein are importantdifferences that distinguish the CD1 and MHC antigen presenting systems.These differences point to a distinct role for CD1 restricted T-cells incell-mediated immunity.

EXAMPLE 2 A Non-Peptide Antigen is Presented by CD1b

Methods

The CD1b-presented antigen is a nondialyzable macromolecule (data notshown). More antigenic (i.e., T-cell proliferative) activity could beobtained from soluble aqueous sonicates of M. tb. by adding detergentssuch as CHAPS or octylglucoside during the sonication (see above). Thisresult suggests that the antigen is hydrophobic.

In order to characterize the chemical nature of the antigens presentedby CD1, mycobacterial antigens were purified from the nonpathogenic M.tb. strain H37Ra (Difco) and M. fortuitum (a rapidly growing strain thatalso contains antigenic activity). Bacteria were either commerciallyavailable (M. tb. H37Ra, Difco) or grown and harvested (M. fortuitum),sonicated and subjected to sequential fractionation protocols andanalyzed for biological activity. All fractions generated were testedfor their ability to stimulate the DN T-cell line DN1 in a 5 dayproliferation assay using irradiated, GM-CSF- and IL-4-treated monocytesas APCs and measuring ³H-thymidine incorporation in a 6 hour pulse(Porcelli, S., et al., Nature 360:593–597 (1992)). Cell wall, cellmembrane and cytoplasmic fractions were prepared from either M. tb. orM. fortuitum using a method adapted from published protocols. Hunter, S.W., et al., Journal of Biological Chemistry 265:14065–14068 (1990).Briefly, cells were lyophilized, resuspended in PBS/octylglucoside,sonicated for 20 minutes and subjected to differentialultracentrifugation to produce cytosolic, membrane, and cell wallfractions. The cell wall pellets were further purified by a differentialsucrose gradient. Characteristic structural features of the threefractions were confirmed by negative staining with electron microscopy.The majority of the bioactivity for the DN1 cell line was present withinthe cell wall fraction (data not shown).

To directly assess whether the CD1b-restricted antigen is a protein, aseries of protease digestions of the antigen were performed. Using avariety of endopeptidases with either limited amino acid specificity(chymotrypsin (hydrophobic residues), trypsin (lys, arg), and V-8(acidic)), or broad amino acid recognition (subtilisin, proteinase K,pronase), sonicates of either M. tb. or M. fortuitum were digested andthen assayed for the ability to induce T-cell proliferative responses.As a control, a DR7 restricted, CD4⁺ T-cell clone DG.1, derived in thislaboratory which recognizes a determinant in mycobacterial PPD (purifiedprotein derivative) was also tested. Analysis by SDS-PAGE and subsequentsilver stain demonstrated that digestion with V8 protease, proteinase K,pronase E or subtilisin degrades the proteins contained in mycobacterialantigen preparations (data not shown).

Results

The M. tuberculosis antigen recognized by DG.1, a representative CD4⁺MHC Class II restricted T-cell line, is rendered ineffective bytreatment with V8 protease, proteinase K, or trypsin (FIG. 6). As shownin FIG. 6, DG.1 cells proliferated strongly in response to the mockdigest of the mycobacterial sonicate, but with the exception ofchymotrypsin, all of the other protease treatments completely abrogatedthe proliferative response.

In contrast, the M. tuberculosis and M. fortuitum antigens presented toline DN1 by CD1b is unaffected by these broadly reactive proteases(FIGS. 7 and 8, respectively). The mycobacterial antigen presented byCD1b is fundamentally different than that presented by MHC Class I andII antigen presenting molecules. It is well established that MHCmolecules bind and present peptide antigens of about 8–9 amino acids forclass I and 13–25 amino acids for class II. Because this CD1b-presentedantigen is a macromolecule which is protease resistant, it is unlikelyto be a peptide. Thus, the CD1 system is the first known antigenpresentation system which present foreign substances other than peptidesto α:β TCR⁺ T-cells.

EXAMPLE 3 Purification of a CD1b-Presented Antigen from M. tuberculosis

Methods

M. fortuitum bacteria were grown in liquid culture to stationary phaseand collected by centrifugation, sterilized by stream autoclaving (250°C., 18 p.s.i.) and lyophilized. Desiccated M. tb. (strain H37Ra, Difco)or M. fortuitum bacteria were suspended in phosphate buffered saline(200 mg bacteria per 5 ml PBS), and the bacterial suspension wassonicated with a probe sonicator to disrupt the cells. The resultingsonicate was extracted with organic solvents using a Folch based 2 phaseextraction system (chloroform/methanol/water) which quantitativelyextracts mycobacterial lipids into an organic phase. Goren, M. B., andBrennan, P. J., Mycobacterial Lipids: Chemistry and Biologic Activitiesin Tuberculosis, 1979. The sonicate was combined with three volumes of achloroform:methanol (2:1 v/v) solution in a glass container, and themixture was vigorously shaken at room temperature for 24 hours. Thephases of the mixture were separated by centrifugation at 800 g, and theorganic phase was collected and transferred into a glass boiling flask.Each fraction was then dried by rotary evaporation (organic phase) orlyophilized (aqueous phase and interface). After evaporation, theorganic phase left a thin film of waxy material on the surface of theflask. In order to prepare material to be tested in T-cell proliferationassays, aliquots of fractions were reconstituted as liposomes by theaddition of water (20 ml per 200 mg of bacteria in original sonicate)followed by sonication in a water bath sonicator. The resulting crudesuspension was then forced repeatedly through a 0.1 nm filter membranein order to create a liposome suspension of uniform size. Alternatively,T-cell media with 10% fetal calf serum was added to the dried fractionand sonicated without additional preparation.

For further purification, the material extracted from M. tuberculosis asdescribed above was dissolved in hexane and applied to a column ofSilicic Acid. Eluting with organic solvents of increasing polarity oversilica columns achieves separation of lipids based on their polarity.The most polar lipids such as phospholipids bind the strongest to thesilica column and elute last, while glycolipids generally bind lesstightly and elute earlier. Neutral lipids such as triglycerides orsterols bind the weakest and therefore elute first.

Small open solid phase extraction (SPE) columns (BakerBond, JT Baker)were preferred because of the ability to process many samplessimultaneously. A silica based “bonded” column (covalently linked) withcyano (CN) functional groups was used to fractionate the organicextracts of M. tb. The organic phase of the choloroform/methanol extractof bacteria was dried and resuspended in hexane. The equivalent of 5.3mg of desiccated bacteria in 200 μl hexane was loaded onto a 0.5 gram CNSPE column. The column was washed with hexane and then with 25% (v/v)chloroform in hexane. Next, the bioactive fraction was eluted with 85%(v/v) chloroform in hexane with over 100% recovery of bioactivity.Analyzing the active fraction on silica based TLC plates, according tothe method of Kupke and Zeugner (Christie, W. W., Lipid Analysis, p.117, Pergamon Press, Oxford, U.K., 1982)), only two major species oflipid were visualized with cupric acetate, corresponding to free fattyacids and mycolic acids (data not shown). This result reflects a markedpurification from the starting organic material.

Proliferation assays were harvested on day 2 (DG.SF68), day 3 (DG.1) orday 5 (DN1). DG.SF68 is a Vγ2Vδ2 T-cell clone derived in this lab (PNASin press, CM). APCs were GM-CSF- and IL-4-treated monocytes (DN1) orPBMC (DR7⁺) (DG.1), or untreated PBMC (DG.SF68). Cytolytic assays aredisplayed as % specific lysis and were performed as described. Porcelli,S., et al., Nature 341:447–450 (1989). Data shown (FIG. 9) are with aneffector to target ratio of 50:1 and with M. tuberculosis antigen at adilution of 1:20.

Results

The relevant antigen of M. tuberculosis is isolated from a commercialpreparation of strain H37Ra (Difco) by extraction into a mixture ofchloroform and methanol as described above. Although the interfacecontains greater than 95% of the proteins, 100% of the CD1b-restrictedantigenic activity (i.e., ability to induce an α:β TCR DN T-cellproliferative response) of the mycobacterium extracts into the organicphase (FIG. 9 a). This strongly supports the original conclusion of thenonpeptide nature of the relevant bacterial antigen. In contrast, aconventional MHC class II restricted antigen recognized by DN γ:δ TCR⁺T-cells was located in the phase interface between the aqueous andorganic phases (FIG. 9 b). In contrast, in four independent antigenpreparations, the CD1b-restricted antigen quantitatively partitionedinto the organic phase. Results of transfectant cytolytic assays of thephases confirmed that the CD1b-presented antigen is present in theorganic phase (FIG. 10).

Under these conditions, 100% of the mycobacterial CD1-presentedantigenic activity was quantitatively recovered after CN SPEchromatography.

In addition, the organic phase extraction served as an excellentpurification step and the organic phase was used as a starting materialfor subsequent chromatography. An alternative and somewhat more generalprocedure to purify the antigen for subsequent chromatography is tosaponify whole or sonicated bacteria and extract with an acidifiedsolution of hexanes. Further purification of the antigen is obtainedusing Silicic Acid chromatography as described above.

EXAMPLE 4 Mycolic Acid is a Mycobacterial CD1b-Presented Antigen

Given the above results, and other preliminary data suggesting that theT-cell stimulatory activity co-chromatographed on CN modified silicaHPLC columns with preparations of free fatty acid acyl chains (data notshown), it seemed plausible that the CD1b-presented antigen is a uniquemycobacterial lipid, possibly a mycolic acid.

Mycobacteria contain an extraordinary proportion of lipids, amounting to40% of the dry weight of bacillus and 60% of the cell wall; the mycolicacids are perhaps the most numerous and diverse members of mycobacteriallipids. Goren, M. B., and Brennan, P. J., Mycobacterial Lipids:Chemistry and Biologic Activities in Tuberculosis, 1979. Mycolic acidsare α-branched, β-hydroxy fatty acids forming a unique set of structuresthat are found in mycobacteria and related bacterial species. Wolinsky,E., “Mycobacteria,” Chapter 37 in Microbiology: Including Immunology andMolecular Genetics, 3rd Ed., Davis, B. H., ed., Harper & Row,Philadelphia, 1980.

Mycolic acids are principally found in the cell wall, esterified toarabinogalactan polymers linked to the core peptidoglycan (McNeil, M.R., and Brennan, P. J., Res. Microbiol. 142:451–563 (1991); Besra, G.S., Biochemistry 30:7772–7777 (1991); McNeil, M., et al., Journal ofBiological Chemistry 266:13217–13223 (1991)) and can be released byeither alkaline or acid hydrolysis (saponification). Minnikin, D. E.,“Mycolic acids” in CRC Handbook of Chromatography: Analysis of Lipids,Murhergee, K. D., and Weber, N., eds., CRC Press, 1993. Mycolic acidsare the major component of the lipid coat surrounding the organism,giving the organism its hydrophobic surface and characteristic acid faststaining. Goren, M. B., and Brennan, P. J., Mycobacterial Lipids:Chemistry and Biologic Activities in Tuberculosis, 1979.

Unlike eukaryotic and bacterial fatty acids, which range in size fromC₁₂–C₂₄, mycolic acids of Mycobacteria range in size from C₆₀–C₉₀.Minnikin, D. E., “Lipids: Complex Lipids, their Chemistry, Biosynthesisand Roles” in The Biology of Mycobacteria, Vol. 1, Ratledge, C., andSanford, J., eds., Academic Press, London, 1982. Mycolic acids, incontrast to the straight chain fatty acids, have a branched alkyl groupat the α carbon and a hydroxyl group at the β carbon. Goren, M. B., andBrennan, P. J., Mycobacterial Lipids: Chemistry and Biologic Activitiesin Tuberculosis, 1979; Minnikin, D. E., “Lipids: Complex Lipids, theirChemistry, Biosynthesis and Roles” in The Biology of Mycobacteria, Vol.1, Ratledge, C., and Sanford, J., eds., Academic Press, London, 1982;Takayama, K., and Qureshi, N., “Structure and Synthesis of Lipids” inThe Mycobacteria: A Sourcebook, Part A, Kubica, G. P., and Wayne, L. G.,eds., Marcel Dekker, New York & Basel, 1984. The main long alkyl chainof the mycolic acid (the so-called mero group) is heterogenous both inlength and in attached functional groups. In addition to alkene groups(double bonds), the functional groups of mycolic acids include methoxyl,keto, lone methyl barances, ethylenic and cyclopropanoid groups.Minnikin, D. E., “Lipids: Complex Lipids, their Chemistry, Biosynthesisand Roles” in The Biology of Mycobacteria, Vol. 1, Ratledge, C., andSanford, J., eds., Academic Press, London, 1982. The large array offunctional groups available to mycolic acids, their variable chainlength, and their heterogeneity among strains, allow mycolic acids toachieve a potentially large degree of antigenic variation similar tothat provided by peptides with heterogeneity among amino acid sidechains. Thus, these lipid molecules may have an immunological relevancenot previously appreciated. For each mycobacterial species adistinguishable fingerprint exists based on the patterns of mycolic acidmolecules present. Such patterns have been determined for individualspecies by thin layer chromatography (TLC). Minnikin, D. E., “Lipids:Complex Lipids, their Chemistry, Biosynthesis and Roles” in The Biologyof Mycobacteria, Vol. 1, Ratledge, C., and Sanford, J., eds., AcademicPress, London, 1982; Dobson, G., et al., Chemical Methods in BacterialSystematics, Academic Press, 1985; Valero-Guillen, P. L., et al.,Journal of Applied Bacteriology 59:113–126 (1985)), gas chromatography(GC) (Valero-Guillen, P. L., et al., Journal of Applied Bacteriology59:113–126 (1985); Athalye, M., et al., Journal of Applied Bacteriology58:507–512 (1985); Luquin, M., et al., Journal of Clinical Microbiology29:120–130 (1991)) and by high pressure liquid chromatography (HPLC).Qureshi, N., et al., Journal of Biological Chemistry 253:5411–5417(1978); Qureshi, N., et al., Journal of Biological Chemistry 255:182–189(1980); Butler, W. R., et al., Journal of Clinical Microbiology29:2468–2472 (1991); Butler, W. R., and Kilburn, J. O., Journal ofClinical Microbiology 28:2094–2098 (1990).

Methods

In order to determine if the CD1-presented antigen described above is amycolic acid, an HPLC method that separates mycolic acids on C18 reversephase column chromatography was used to prepare mycolic acids. Butler,W. R., et al., Journal of Clinical Microbiology 23:182–185 (1986);Butler, W. R., et al., Journal of Clinical Microbiology 26:50–53 (1988);Floyd, M. M., et al., Journal of Clinical Microbiology 30:1327–1330(1992). Reversed phase chromatography separates acyl chains primarily onthe basis of the length of the acyl chain or “carbon number,” thus it isrelatively easy to achieve good separation between free fatty acids andmycolic acids which are much larger.

This HPLC method requires an initial saponification of the sample,followed by derivitization of the fatty acids or mycolic acids with theultraviolet (OD₂₅₄) absorbing compound p-bromophenacylbromide, whichattaches to the carboxyl terminus of an acyl chain. In preliminaryexperiments, we determined that the process of derivatizing thebacterial fraction with p-phenacylbromide destroyed bioactivity.However, the CD1b-restricted antigenic activity could then be recoveredby saponification with methanolic KOH, a process which frees thecarboxyl end of the acyl chain by cleaving off the phenacylbromide group(as assayed by OD₂₅₄ on HPLC). This result indicates that the acyl chainmust be cleaved in order to achieve a form which is presentable by CD1positive APCs and/or that a free carboxyl group is critical forpresentation of the CD1b-restricted antigen. This is additional evidencethat the antigen comprises an acyl chain.

The SPE CN column purified preparation (Example 3) was used as astarting material for the C18 chromatography. Samples were saponifiedwith methanolic KOH and derivatized with the UV absorbing groupbromophenacyl bromide. The active fraction was run on a C18 column(Alltech Nucleosil C18 5 μm, 25 cm×4.6 mm) using a linear gradient of 30to 90% methylene chloride in methanol over 50 minutes at 1 ml/min. Usingas a reference a C₉₀ internal standard (Ribi), the resultingchromatogram (FIG. 12, panel a, upper portion) has a pattern comparableto published results. Floyd, M. M., et al., Journal of ClinicalMicrobiology 30:1327–1330 (1992). Fractions were resuspended in completemedia with 10% FCS and tested at a 1:17 dilution relative to theoriginal sonicate volume.

Results

Bioactivity, assayed by the T-cell proliferative response assay, wasfound to comigrate with early peaks in the region of the mycolic acids(FIG. 12 a). To confirm that mycolic acid is presented by CD1b, analternative source of mycolic acids, purified cord factor (trehalosedimycolate; FIG. 11) was tested. Upon saponification, purified trehalosedimycolate from either M. tb. (from Sigma) or M. kansasii (gift ofPatrick Brennan) stimulated the proliferation of the T-cell line DN1(FIG. 12 b). However, no stimulation was achieved by material preparedfrom saponified trehalose dibehanate, a synthetic derivative of cordfactor which contains two C₂₂ fatty acid chains, rather than mycolicacids, that are liberated by saponification. This strongly argues thatmycolic acid, not trehalose (which is present in each of the samples)nor fatty acids, is the antigen presented by CD1b to the double negativeα:β TCR T-cell line DN1. A HPLC analysis of the saponified Sigma cordfactor was then performed, and again the bioactivity was located infractions corresponding to the early mycolic acid peaks (FIG. 12 c).Together, the above data demonstrate that the CD1b restrictedmycobacterial antigen recognized by the T-cell line DN1 is a species ofmycolic acid.

Mycobacteria are known to be highly effective adjuvants. Aldovini, A.,and Young, R. A., Nature 351:479–482 (1991). One source of theCD1b-presented antigen, mycolic acid, used herein is trehalosedimycolate (i.e., Mycobacterial cord factor), which has been shown toenhance antibody formation (Bekierkunst, A., et al., J. Bacteriol.100:95–102 (1969); Bekierkunst, A., et al., Infection and Immunity4:245–255 (1971); Bekierkunst, A., et al., Infection and Immunity4:256–263 (1971)) and to stimulate nonspecific immunity to bacterialinfections (Parant, M., et al., Infect. Immun. 20:12–19 (1978)) andtumors (Bekierkunst, A., et al., Infection and Immunity 10:1044–1050(1974).

In order to ensure that the purified antigenic activity contains a bonafide antigen and not a nonspecific mitogen, we looked at the specificityof the T-cell proliferative response to crude M. tb. preparations andHPLC-purified mycolic acid from M. tb. and saponified cord factor. Totalsonicates of M. tb. (FIG. 14, upper panel) contain antigen recognized bythe MHC class II restricted CD4⁺ α:β TCR⁺ T-cell line DG.1, as well asan antigen recognized by the CD1b-restricted T-cell line DN1 and theCD1c-restricted T-cell line DN6 (see Example 5, below). However,HPLC-purified mycolic acid from either M. tb. (FIG. 14, middle panel) orfrom saponified cord factor (FIG. 14, lower panel) contains antigensrecognized only by CD1b-restricted DN1 T-cells. This specificity is alsodemonstrated in the transfectant cytolytic to assay (FIG. 13). TheCD1b-restricted response of DN1 was blocked by anti-CD1b antibodies, butwas not affected by antibodies to MHC class I or II (FIG. 15, upperpanel).

EXAMPLE 5 Antigen Presentation by CD1c

In addition to the presentation of antigens by CD1b disclosed in Example1, CD1c molecules also present antigens. A separate CD4⁻8⁻ α:β TCR⁺T-cell line, derived by repeated stimulation with monocytes treated withGM-CSF and IL-4 (to induce CD1 expression) and M. tuberculosis antigenswas isolated and named DN2 (also referred to as 8.23.DN1). Proliferationof DN2 is completely inhibited by the addition of mAbs to CD1c, but isnot affected by mAbs to CD1b (FIG. 15, lower panel). A cytolytic assayconfirms this result: C1R cells transfected with vector only or withvector encoding CD1a, CD1b or CD1c were preincubated with or without M.tuberculosis sonicate and used as targets in a cytolytic assay. OnlyCD1c⁺ C1R cells are recognized (FIG. 16); recognition is enhanced bypreincubation with the sonicate. Therefore, CD1c molecules present M.tuberculosis antigens to DN2 T-cells.

A second CD1c-restricted CD4⁻8⁻ α:β TCR⁺ T-cell line, derived byrepeated stimulation with monocytes treated with GM-CSF and IL-4 (toinduce CD1 expression) and M. tuberculosis antigens was isolated andnamed DN6. Cytolytic assays (FIG. 17) indicate that DN6 lyses CD1c⁺cells in the presence of M. tb. antigen.

EXAMPLE 6 Characterization of a CD1c-Presented Antigen from M.tuberculosis

The antigen presented to DN6 T-cells by CD1c is not mycolic acid (FIG.14, middle panel). However, chemical characterization of the antigenrecognized by DN6 indicates that the antigen is a complex lipid. When M.tb. sonicates are extracted with chloroform:methanol (as described inExample 3), the antigenic activity is found substantially in both thephase interface and the organic phase (FIG. 18). Antigen recovered fromthe organic phase can be bound to CN SPE columns and eluted (asdescribed in Example 3). These studies demonstrate that the antigen ishydrophobic and has the chromatographic properties of a lipid.

However, unlike the CD1b-restricted mycobacterial antigen, results ofadditional experiments indicate that the DN6-recognized CD1c-presentedantigen is a complex lipid and not a free acyl chain. Saponification ofM. tb. sonicates eliminates the proliferative response of DN6 (FIG. 20).Because saponification breaks ester linkages that connect acyl chains tocarbohydrate backbones, this result suggests that the antigen recognizedby DN6 T-cells is a, or comprises an additional, moiety other than afree acyl chain. Saponification presumably destroys or removes theadditional moiety which may be, e.g., a polysaccharide backbone orbranch point. Thus, in contrast to the recognition of free mycolic acidby the DN1 T-cell line, the DN6 T-cell line recognizes a more complexlipid structure.

It is noteworthy that the CD1-presented antigens recognized by T-celllines DN1 and DN6 are not unique to M. tuberculosis. Rather, the DN1-and DN6-recognized CD1-presented antigens are cross-reactive to antigensfound in many Mycobacterial species tested to date (M. fortuitum, M.avium, M. bovis (BCG) and M. leprae). In the case of CD1c-restrictedrecognition by DN6 T-cells, an antigen is recognized in Corynebacteria(data not shown), a separate but related genera of bacteria whichproduce mycolic acids. Takayama, K., and Qureshi, N., “Structure andSynthesis of Lipids” in The Mycobacteria: A Sourcebook, Part A, Kubica,G. P., and Wayne, L. G., eds., Marcel Dekker, New York & Basel, 1984.Thus, CD1-presented antigens include at least several bacterial lipidantigens; in the case of autoimmunity, CD1-presented antigens includeendogenous lipid antigens.

EXAMPLE 7 CD1-Presented Antigens from M. leprae

In order to derive DN α:β TCR⁺ T-cell lines from leprous lesions,T-cells from such lesions or PBMC were stimulated by culture with CD1⁺APCs and M. leprae. Immunomagnetic depletion removed CD4⁺, CD8⁺ andγ:δ-TCR⁺ T-cells. The ability of remaining CD4⁻8⁻ α:β TCR⁺ T-cells torespond to M. leprae antigens was assessed by ³H-thymidine incorporationusing allogenic CD1+ monocytes as APCs.

Four of six DN α:β TCR⁺ T-cell lines isolated in the above mannerproliferated strongly in the presence of M. leprae and allogenic CD1+APCs, whereas little or no stimulation of growth was detected in thepresence of the allogenic CD1+ APCs alone, and no proliferation inreponse to M. leprae was detected in the presence of APCs not expressingCD1 proteins. Thus, the four DN α:β TCR⁺ T-cell lines respond to, andproliferate in the presence of, CD1-presented antigen from M. leprae.

Two DN α:β TCR⁺ T-cell lines, isolated from leprous lesions anddesignated LDN1 and LDN4, were subjected to further analyses. Todetermine which CD1 molecules (i.e., CD1a, CD1b, Cd1c, etc.) werespecifically responsible for M. leprae antigen presentation, two sets ofexperiments were performed. First, proliferation studies were performedin which CD1⁺ APCs were incubated with antibodies to CD1a, CD1b or CD1c.Second, C1R cells were transformed with genes encoding different CD1molecules and used as target cells in cytolytic assays.

M. leprae-induced proliferation of the LDN1 T-cell line was inhibited bygreater than 63% by anti-CD1c, but anti-CD1a, anti-CD1b and anisotypematched non-CD1-reactive antibody had no effect on theproliferative response to M. leprae (FIG. 20, upper panel). Similarly,LND1 cells lysed CD1c-transformed C1R cells in an M. leprae antigenspecific manner, but did not lyse cells transfected with CD1a or CD1b ormock transfected C1R cells.

A second M. leprae antigen reactive DN α:β TCR⁺ T-cell line derived froma leprous lesion, LND4, was found to be Cd1b restricted. The ability ofthis T-cell line to proliferate in the presence of M. leprae wascompletely inhibited by the addition of anti-CD1b monoclonal antibodies(MAbs) (FIG. 20, lower panel). Similarly, LND4 lysed CD1b-transfectedC1R cells in an antigen specific manner, but did not lyse CD1a or CD1ctransfectants. Furthermore, the ability of LND4 to lyse antigen-pulsedCD1b targets was blocked by anti-CD1b MAbs. In all DN α:β TCR⁺ T-celllines tested, antibodies to MHC class I and class II molecules had noeffect on proliferation in response to M. leprae.

The presence of CD1-bearing cells in leprous lesions was examined byimmunohistological examination of tissue sections using MAbs to CD1(data not shown). These analyses reveal that CD1a+, CD1b+ and CD1c+cells occur in in tuberculoid granulomas to a greater extent than inlepromatous granulomas. Although CD1a was expressed in the epidermis aswell as the dermal granulomas of tuberculoid leprous lesions, CD1b andCD1c were only expressed in dermal granulomas. Furthermore, Il-10, whichis strongly expressed in lepromatous lesions (Yamamura, M., et al.,Science 254:277–279 (1991)) could inhibit the expression of CD1 onmonocytes (data not shown). The correlation of CD1 expression withresistance in leprosy suggests a role for CD1-mediated restriction inthe generation of cell-mediated immunity.

The functional role of DN α:β TCR⁺ T-cells was examined by studyingtheir cytokine secretion patterns. Anti-CD3 stimulation of DN α:β TCR⁺T-cells resulted in the release of interferon-γ (IFN-γ) (median=647pg/ml) in four of the five DN α:β TCR⁺ T-cell lines, but produced littleor no IL-4 (median<20 pg/ml), although one T-cell line produced IL-4 (99pg/ml) but no detectable IFN-γ (<20 pg/ml). IL-5, IL-6 and IL-10 werenot detected in the superantants of anti-CD3-stimulated DN T-cells.These data suggest that the majority of M. leprae-reactive DN α:β TCR⁺T-cells secrete the type 1 cytokine pattern.

EXAMPLE 8 LAM is a CD1b-Presented Antigen from M. leprae

In order to elucidate the biochemical nature of the antigens recognizedby CD1-restricted T-cells, purified cell wall constituents of M. lepraewere prepared and assayed for biological activity. LDN4, in the presenceof CD1 expressing antigen presenting cells, proliferated in response tolipid-containing fractions derived from the cell wall (SolPCW), but notto protein-enriched fractions from the membrane and cytosol that havebeen depleted of lipids (SP-) (FIG. 21). No response was observed toinsoluble fractions of the cell wall (CWC) or to mycolicacid-arabinogalactan-peptidoglycan (mAGP). These data indicate that DNαβ T-cells recognize non-peptide antigens of M. leprae in aCD1-restricted manner.

The reactivities of LDN4 correlated with the presence oflipoarabinomannan (LAM; FIG. 22), and we subsequently found that thisline proliferated to purified LAM. The ability of LDN4 to respond to LAMwas blocked by anti-CD1b antibody (FIG. 23). BDN2, an α:β doublenegative T-cell line which responds to M. leprae in a CD1c restrictedmanner, also responded to LAM. The ability of BDN2 to respond to LAM wasblocked by anti-CD1c antibody (data not shown).

EXAMPLE 9 Derivatives and Analogs of LAM, and CD1-Dependent Uses Thereof

LAM is a nonprotein molecule, one of a class known as “amphiphiles,”possessing both hydrophobic and hydrophilic components (FIG. 22).Hunter, S. W., etal., J. Biol. Chem. 261:12345–12531 (1986); Hunter, S.W., et al., J. Biol. Chem. 265:9272–9279 (1990). The hydrophobic domainis a phosphatidyl inositol linked to a hydrophilic heteropolysaccharidecomposed of a mannan core and branched arabinan side chains with shortmannose oligosaccharide “caps.” Chatterjee, D., et al., J. Biol. Chem.267:6228–6233 (1992); Chatterjee, D., et al., J. Biol. Chem.267:6234–6239 (1992). Protocols for the purification of LAM have beendescribed. Hunter, S. W., et al., J. Biol. Chem. 261:12345–12531 (1986).Fast Atom Bombardment-Mass Spectrometry analysis indicates the virtuallycomplete purity of the M. leprae LAM (>99.9%).

Previously, M. leprae and M. tuberculosis LAM have been shown to havedistinct structures which result in different B-cell epitopes. Prinzis,S., et al., J. Gen. Microbiol. 139:2649–2658 (1993). Therefore, it isconceivable that LAM from different mycobacterial species may possessdistinct T-cell epitopes. It is also noteworthy that gram positivebacteria contain structurally related lipomannans and lipoteichoicacids, so that recognition of these molecules by T-cells may be a partof the immune response directed generally to bacterial pathogens.

To determine the domain(s) of LAM responsible for stimulating DN α:βT-cells, chemical derivatives of LAM were tested for their ability tostimulate DN α:β T-cells. Mild alkaline hydrolysis releases the fattyacid moieties of LAM, while leaving the carbohydrate intact. Thisdeacylated LAM (dLAM) loses the ability to stimulate LDN4 atconcentrations where the native LAM caused striking T-cell proliferation(FIG. 24). In contrast to deacylated LAM, phosphatidylinositol mannoside(PIM) stimulated LDN4 ten-fold more than media alone (FIG. 24). Thesedata suggest that the lipid domain of LAM is required for T-cellstimulation, but that the repeating mannan-arabinan backbone of LAM maynot be required.

Finally, the cross-recognition of LAMs from other mycobacteria wasinvestigated with regard to two DN α:β T-cell lines (FIG. 25). Theleprosy-derived T-cell line LDN4 was stimulated by M. leprae LAM (LepLAM), but not by LAM from a clinical isolate of M. tuberculosis(TBE-LAM) or LAM from a virulent laboratory strain of M. tuberculosis(Rv LAM). These results demonstrate a species specificity of LDN4 for M.leprae LAM. In contrast, T-cell line BDN2 responded to M. tuberculosisLAM (TBE LAM and Rv LAM) as well as to M. leprae LAM (Lep LAM). Takentogether, these results demonstrate that both species-specific andcross-reactive determinants of LAM are recognized by DN α:β T-cells.

EXAMPLE 10 Vaccines Comprising CD1-Presented Antigens

Prior to the present disclosure, lipids were not known to possesspotentially potent specific T-cell-mediated immunogenicity. TheCD1-presented lipid antigens described herein may be used as essentialcomponents of compositions designed to function as vaccines tomycobacterial pathogens. Vaccines comprising CD1-presented antigens maybe administered directly by injection. Alternatively, due to thepresence of CD1 on cells found in the gastrointestinal epithelium(Bleicher, P. A., et al., Science 250:679–682 (1990)), oraladministration of vaccines comprising CD1-presented antigens may beemployed to introduce such vaccines into an animal in need of a vaccinecomprising a CD1-presented antigen.

Vaccines comprising the CD1-presented antigens of the present inventionare formulated according to known methods. Remington's PharmaceuticalSciences, 18th Ed., Gennaro, A. R., ed., Mack, Easton, 1990; ThePharmacologist Basis of Therapeutics, 7th Ed., Gilman, A. G., et al.,eds., MacMillian, New York, 1985. Pharmaceutically acceptablelipid-stabilizing and -solubilizing agents known to those of skill inthe art may be added to vaccines comprising CD1-presented antigens toenhance their effectiveness. Teng, N., et al., PCT published patentapplication WO 91/01750 (Feb. 21, 1991). Nunberg, J. H., U.S. Pat. No.4,789,702 (Dec. 6, 1988).

EXAMPLE 11 Means and Methods for Inhibiting CD1-Restricted AntigenPresentation

The disclosure of CD1 antigen presentation system allows for variousmeans and methods of inhibiting CD1-restricted antigen presentation. Anycomposition which inhibits the processing of CD1-presented antigens,which interferes with the binding of antigen to a CD1 molecule, or whichinterferes with the binding of the CD1; antigen complex to a TCRmolecule, will inhibit CD1-restricted presentation of antigen. Suchcompositions, called CD1 blocking agents, are useful for controllingundesired T-cell-mediated immunity which occurs, e.g., in autoimmunediseases. Oksenberg, J. R., et al., J. Neurol. Sci. 115 (Suppl.):S29–S37(1993).

CD1 blocking agents include, but are not limited to, (1) a purified CD1molecule, or a synthetic derivative thereof, capable of binding aCD1-presented antigen and preventing the antigen's interaction with CD1displayed on APCs; (2) a purified TCR polypeptide, or a syntheticderivative thereof, capable of binding to a CD1-antigen complex on aCD1⁺ APC and preventing the complex's interaction with T-cell receptors;(3) an antigen antagonist comprising a chemically-modified CD1-presentedantigen, or a synthetic derivative of a CD1-presented antigen; (4) apurified CD1-antigen complex, or a synthetic derivative thereof, capableof binding to a T-cell receptor that recognizes a CD1-antigen complex ona CD1⁺ APC and preventing the T-cell receptor's interaction withCD1-antigen complexes; (5) an antibody which binds a CD1 molecule andthus prevents the interaction of the CD1 molecule and a CD1-presentedantigen; (6) a polyclonal or monoclonal antibody which binds aCD1:antigen complex and thus prevents the interaction of the CD1:antigencomplex and its cognate TCR; (7) a polyclonal or monoclonal antibodywhich binds a TCR that recognizes a CD1-presented antigen and thusprevents the interaction of the TCR with its cognate CD1:antigencomplex; and (8) a composition which blocks an essential step in thepathway by which CD1-presented antigens are processed prior to beingdisplayed.

The preceding Examples contain exemplars of CD1blocking agents asfollows.

CD1 blocking agents of type (5) are represented by monoclonal antibodyWM25 to CD1b, which specifically inhibits CD1b-restricted antigenpresentation (FIG. 15, upper panel) and monoclonal antibody 10C3 toCD1c, which specifically inhibits CD1c-restricted antigen presentation(FIG. 15, lower panel). A skilled artisan can used the methods describedherein to isolate antibodies which act as CD1 blocking agents of type(6) or (7), and those of skill in the art know how to formulateantibodies for therapeutic purposes. A Critical Analysis of AntibodyTherapy in Transplantation, Burlington, W. J., ed., CRC Press, BocaRaton, 1992.

CD1 blocking agents of type (8) are represented by chloroquine (FIG. 5c), which inhibits a step in the processing of CD1b-restricted antigens.Methods of formulating and administering chloroquine are known to thoseof skill in the art. Webster, L. T., “Drugs Used in the Chemotherapy ofProtozoal Infections,” Chapters 41 and 42 in Goodman and Gilman's ThePharmacological Basis of Therapeutics, 8th Ed., Gilman, A. G., et al.,eds., Pergamon Press, New York, 1990. Although chloroquine also inhibitsMHC-restricted antigen presentation, those skilled in the art can, usingthe methods disclosed herein, identify and/or isolate compositions whichspecifically inhibit processing of CD1-restricted antigens.

Blocking agents of type (3), i.e., antigen antagonists, may be derivedfrom CD1-restricted antigens isolated by the methods of the invention.Variants of MHC-restricted peptide antigens, binding with weakeraffinities than the original peptide antigen, act as antagonists formature T-cells in MHC-restricted antigen presentation. Wraith, D. C., etal., Cell 59:247–255 (1989); Smilek, D. E., et al., Proc. Natl. Acad.Sci. (USA) 88:9633–9637 (1991). In like fashion, CD1-presented antigensare isolated by the methods of the invention and are chemicallymodified, according to standard techniques, in order to generatenon-antigenic or weakly antigenic CD1-presented antigen derivatives. Forexample, mycolic acid derivatized with p-bromophenacylbromide isnon-antigenic (Example 4). Antigen antagonists are identified asCD1-presented antigen derivatives which inhibit or prevent the T-cellproliferative or CD1 transfectant cytolytic responses which occur whenonly the original, unmodified CD1-presented antigen is present (Example1).

Blocking agents of type (2), i.e., TCR derivatives which block theinteraction of the antigen:CD1 complex with the TCR molecules onT-cells, may be prepared from the disclosure by those skilled in theart. DNA molecules encoding TCR polypeptides displayed by a T-cell linethat recognizes a CD1-presented antigen of the invention are isolatedaccording to methods known in the art. Oskenberg, J. R., et al., Proc.Natl. Acad. Sci. (USA) 86:988–992 (1989); Oksenberg, J. R., et al.,Nature 345:344–346 (1990) and erratum, Nature 353:94 (1991); Uematsu,Y., et al., Proc. Natl. Acad. Sci. (USA) 88:534–538 (1991); Panzara, M.A., et al., Biotechniques 12:728–735 (1992); Uematsu, Y., Immunogenet.34:174–178 (1991). The DNA sequence is converted into a polypeptidesequence, and the portion of the polypeptide sequence that correspondsto the antigen-binding variable region of a TCR polypeptide is used todesign synthetic oligopeptides that bind CD1:antigen complexes on APCs,thereby inhibiting antigen presentation. Oligopeptides are chemicallysynthesized according to standard methods (Stewart and Young, SolidPhase Peptide Synthesis, Pierce Chemical Co., Rockland, Ill., 1985) andpurified from reaction mixtures by reversed phase high pressure liquidchromatography (HPLC). Pilot trials of treatment of humans sufferingfrom an autoimmune disease, MS, with peptides derived fromMHC-restricted α:β TCR molecules is currently underway. Oksenberg, J.R., et al., J. Neurol. Sci. 115 (Suppl.):S29–S37 (1993). TCR-derivedpeptides which function as CD1 blocking agents are identified asTCR-derived peptides which inhibit or prevent the T-cell proliferativeor CD1 transfectant cytolytic responses which occur in the presence ofCD1-presented antigen (Example 1).

The assays for CD1-presented antigens described herein may be used toscreen molecular libraries for CD1 blocking agents. Libraries ofmolecularly diverse compositions are prepared by chemical, biochemicaland/or biotechnological means. Such libraries include combinatoriallibraries of synthetic peptides (Houghten, R. A., et al., BioTechniques13:412–421 (1992)) and fusion protein libraries prepared by recombinantDNA technology, e.g., phage display libraries. Koivunen, E., et al., J.Biol. Chem. 268:20205–20210 (1993). The libraries are screened for thepresence of members which inhibit or prevent the DN T-cell proliferativeand/or CD1 cytolytic responses described herein. CD1 blocking membersare isolated from the library according to techniques known in the artand appropriate for the type of library employed. Lowman, H. B., et al.,Biochemistry 30:10832–10838 (1991); Felicia, F., et al., J. Mol. Biol.22:301–310 (1991); Dandekar, T., et al., Neurochem. Int. 7:247–253(1985); Owens, R. A., et al., Biophys. Res. Commun. 181:402–408 (1991).

In order to detect a CD1 blocking agent in a sample, assays forCD1-presented antigen are performed in duplicate. The first (control)assay is a T-cell proliferative or cytolytic assay performed essentiallyas described in Example 1. The second assay is substantially the same asthe first assay except that the second assay additionally contains asample suspected of containing a CD1 blocking agent. The presence of CD1blocking agents in the sample correlates with a T-cell proliferative orcytolytic response in the second assay that is significantly less thanthat measured in the first assay.

EXAMPLE 12 T-cell Reactivity to CD1-Presented Antigens

As described in Example 1, T-cell reactivity to CD1-restricted antigenswas initially found among a subset of T-cells, i.e., CD4⁻8⁻ TCR αβ⁺T-cells. However, additional examples of CD1-restricted foreign antigenrecognition of microbial antigens is also found in CD8⁺ TCR αβ⁺ T-cells,as well as among TCR γδ⁺ T-cells.

CD1 Restricts the Response of TCR γδ⁺ T-cells to an Antigen

Human peripheral mononuclear cells were obtained from a leukopak. Afterdepletion of the monocyte population by plastic adherence, TCR γδ⁺T-cells were isolated using magnetic cell sorting (MACS) as previouslydescribed ((Schittek, B. and Rajewsky, K., Nature 346:749–751 (1990)).Briefly, PBMC were labelled with biotinylated anti-γδ TCR monoclonalantibody (TCRδ1), followed by staining with FITC-Avidin (CALTAG) andbiotinylated beads (Miltenyi Biotec, Sunnyvale, Calif.). Cells were thenpositively selected in a strong magnetic field through a high capacity(3×10⁷ cells) column (Miltenyi Biotec, Sunnyvale, Calif.). The purity ofthe cells was assessed by FACS immediately after purification. Startingfrom 2×10⁹ PBMC, this procedure typically allows one to obtain 2×10⁷ TCRγδ⁺ T-cells with >98% purity.

After three days in vitro without specific stimulation, the purified TCRγδ⁺ T-cells were cultured with autologous GM-CSF- and IL-4-treated CD1⁺monocytes at a 1:1 ratio in 24 well plates (Costar, Cambridge, Mass.)(plating 2×10⁶ T-cells per well). The antigen used to stimulate thecells was an organic extract of Mycobacterium tuberculosis (as describedin Examples 2 and 3) at a 1/3,750 dilution. Briefly, achloroform/methanol extraction based on Folch's method, which partitionsmost lipids into the organic phase and protein into the aqueous phaseand interface, was used. Recombinant Interleukin-2 (rIL-2) was added(0.3 nM) after 4 days of culture and the cells were restimulated withantigen every 12 days.

Three rounds of stimulation led to the isolation of the CD1b-restrictedTCR γδ⁺ T-cell line called OGD1. Although the purified TCR γδ⁺ T-cellpopulation from which this T-cell line was isolated predominantlyexpressed the Vδ2 gene product (90%), FACS analysis of OGD1 with themonoclonal antibody TCR δ1 (γδTCR), δTCS1 (Vδ1 gene product) and BB3(Vδ2 gene product) (available from T Cell Diagnostics, Cambridge, Mass.)demonstrated that the isolated T-cells expressed predominantly the Vδ1gene product (FIG. 26A). This result suggests that this CD1 mediatedstimulation is different than all previous demonstrated examples ofstimulation of TCR γδ⁺ T-cells by bacterial antigens, which invariablyinvolve expansion of Vγ2Vδ2 T-cells.

The restriction of the OGD1 cell line by CD1b was confirmed usinganti-CD1b monoclonal antibody which blocked cytotoxicity of antigenpulsed CD1⁺ monocytes (FIG. 27) and also blocked specific proliferationof the OGD1 cell line to mycobacterial antigens present in the organicphase of chloroform/methanol extracted mycobacteria (FIG. 28). Furtheranalysis of the fine specificity of this cell line demonstrated thatOGD1 recognized saponified cord factor (SIGMA, St. Louis, Mo.) (i.e.,mycolic acid, a lipid component of the mycobacterial cell wall) (FIG.28).

CD8⁺ TCR αβ⁺ T-cells Recognize Antigens Presented by the CD1 Molecule

In order to determine if the CD1 molecule could serve as an antigenpresenting molecule for CD8⁺ TCR γδ⁺ bearing T-cells, experimentssimilar to those described above were performed using purifiedperipheral blood CD8⁺ T-cells instead of TCR γδ⁺ T-cells. T-cells werepurified with magnetic beads conjugated with anti-CD8 monoclonalantibody (Miltenyi Biotec, Sunnyvale, Calif.) and purity was assessed byFACS after subsequent staining with FITC conjugated monoclonal antibodyOKT8 (anti-CD8α). The culture protocol was similar to that for TCR γδ⁺T-cells. After three stimulations of these CD8⁺ T-cells with CD1⁺monocytes pulsed with the organic extract of Mycobacterium tuberculosis,a cell line was obtained which was named OAB8. FACS analysis withFITC-conjugated anti-CD8 α-chain (OKT8) and anti-CD8 β-chain (2ST8-5H7)monoclonal antibodies showed that the cell line was homogeneouslypositive for expression of the CD8 αβ heterodimer. Staining with TCRαβ-specific monoclonal antibody BMA031 showed that cell line OAB8 washomogenously positive for TCR αβ. Proliferation assays confirmed thespecificity of the OAB8 cell line to the organic extract of mycobacteriaand showed that OAB8 recognized purified mycobacterial mycolic acid(FIG. 29). Moreover, antibody blocking experiments demonstrated that theOAB8 cell line was restricted by the CD1c molecule (FIG. 30), asanti-CD1c monoclonal antibody blocked its response whereas antibodies toCD1a, CD1b and MHC class I molecules had no effect.

These data indicate that in addition to the reactivity of CD4⁻8⁻ TCR αβ⁺bearing T-cells to CD1-restricted microbial antigens, other major T-cellpopulations are CD1 restricted. Because CD8⁺ TCR αβ⁺ bearing T-cellsaccount for approximately 35% of all circulating T-cells and TCR γδbearing T-cells account for another 5% of all T-cells, the potentialmagnitude of the CD1-restricted immune response is likely to besignificant in a variety of immunological response and syndromes.Moreover, since these T-cell populations are known to secrete an arrayof cytokines and to mediate cytolytic activity, the effectorcapabilities of CD1-restricted CD8⁺ TCR αβ⁺ and TCR γδ⁺ T-cells specificfor nonpeptide antigens may play a critical role in host defense.

Incorporation by Reference

The entire text of all publications cited herein are entirelyincorporated herein by reference.

1. A method for inhibiting presentation of a CD1b restricted antigen byCD1b⁺ cells, comprising the step of: contacting cells displaying a CD1bmolecule with a CD1b blocking agent which inhibits CD1b-restrictedantigen presentation, said agent comprising an antibody that binds tothe CD1b molecule and blocks the interaction of said CD1b molecule withthe CD1b-restricted antigen.
 2. A method for inhibiting presentation ofa CD1b restricted antigen by CD1c⁺ cells, comprising the step of:contacting cells displaying a CD1c molecule with the CD1c blocking agentwhich inhibits CD1c-restricted antigen presentation, said agentcomprising an antibody that binds to the CD1c molecule and blocks theinteraction of said CD1c molecule with the CD1c-restricted antigen,wherein the contacting occurs in vivo.
 3. A method according to claim 2,wherein said CD1c molecule presents a CD1c-restricted autoimmuneantigen.
 4. The method of claim 1, wherein the contacting occurs invivo.
 5. A method for inhibiting presentation of a CD1a restrictedantigen by CD1a⁺ cells, comprising the step of: contacting cellsdisplaying a CD1a molecule with a CD1a blocking agent which inhibitsCD1a-restricted antigen presentation, said agent comprising an antibodythat binds to the CD1a molecule and blocks the interaction of said CD1amolecule with the CD1a-restricted antigen.
 6. A method of claim 5,wherein said CD1a molecule presents a CD1a-restricted autoinmiuneantigen.
 7. The method of claim 5, wherein the contacting occurs invivo.
 8. A method for inhibiting presentation of a CD1d restrictedantigen by CD1d⁺ cells, comprising the step of: contacting cellsdisplaying a CD1d molecule with a CD1d blocking agent which inhibitsCD1d-restricted antigen presentation, said agent comprising an antibodythat binds to the CD1d molecule and blocks the interaction of said CD1dmolecule with the CD1d-restricted antigen.
 9. A method of claim 8,wherein said CD1d molecule presents a CD1d-restricted autoimmuneantigen.
 10. The method of claim 8, wherein the contacting occurs invivo.
 11. A method for inhibiting CD1c-restricted antigen presentationby CD1c⁺ cells, comprising the step of: contacting cells displaying aCD1c molecule with a CD1c blocking agent which inhibits CD1c-restrictedantigen presentation, said agent comprising an antibody, wherein thecontacting occurs in vivo.
 12. The method of claim 11, wherein theantibody binds to a CD1c:antigen complex.
 13. A method for inhibitingCD1a-restricted antigen presentation by CD1a⁺ cells, comprising the stepof: contacting cells displaying a CD1a molecule with a CD1a blockingagent which inhibits CD1a-restricted antigen presentation, said agentcomprising an antibody.
 14. The method of claim 13, wherein the antibodybinds to a CD1a:antigen complex.
 15. A method for inhibitingCD1d-restricted antigen presentation by CD1d⁺ cells, comprising the stepof: contacting cells displaying a CD1d molecule with a CD1d blockingagent which inhibits CD1d-restricted antigen presentation, said agentcomprising an antibody.
 16. The method of claim 15, wherein the antibodybinds to a CD1d:antigen complex.